WO2017027622A1 - Analyse optique de particules et de vésicules - Google Patents

Analyse optique de particules et de vésicules Download PDF

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Publication number
WO2017027622A1
WO2017027622A1 PCT/US2016/046401 US2016046401W WO2017027622A1 WO 2017027622 A1 WO2017027622 A1 WO 2017027622A1 US 2016046401 W US2016046401 W US 2016046401W WO 2017027622 A1 WO2017027622 A1 WO 2017027622A1
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Prior art keywords
probe
particle
optical
particles
optical signal
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PCT/US2016/046401
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English (en)
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John P. Nolan
Erika DUGGAN
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Scintillon Institute For Biomedical And Bioenergy Research
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Priority to EP16754371.9A priority Critical patent/EP3335028A1/fr
Publication of WO2017027622A1 publication Critical patent/WO2017027622A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/06—Investigating concentration of particle suspensions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1429—Signal processing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • G01N15/1459—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/06—Investigating concentration of particle suspensions
    • G01N15/075—Investigating concentration of particle suspensions by optical means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/06—Investigating concentration of particle suspensions
    • G01N2015/0687—Investigating concentration of particle suspensions in solutions, e.g. non volatile residue
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1029—Particle size
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1486—Counting the particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1488—Methods for deciding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1493—Particle size
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks

Definitions

  • the technology relates in part to optical methods for analyzing particles and vesicles, including membrane vesicles such as liposomes and extracellular vesicles.
  • Optical methods for detecting particles and/or determining their identity, number, size or origin have long been in use.
  • the staining of particles using optically detectable labels generally must be accompanied by one or more washing and/or centrifugation procedures to remove background interference from unbound label.
  • ultracentrifugation can lead to inefficiencies as well as inaccuracies in the analyses, especially when analyzing small volumes of sample, due to partial loss of particles during the separation.
  • EVs extracellular vesicles
  • ectosomes biological membrane vesicles that are released from cell surfaces (ectosomes), internal stores (exosomes) or as a result of apoptosis or cell death
  • ectosomes biological membrane vesicles that are released from cell surfaces
  • exosomes internal stores
  • cell death often provide incorrect estimates of their size and concentration when the vesicles are
  • nanovesicles due to dim light scatter. Further, detection of the EVs often is triggered by coincidence, i.e. , simultaneous detection of the presence of more than one EV in the flow cytometer measurement volume, leading to incorrect concentration, size and fluorescence estimates.
  • Some optical methods such as nanoparticle tracking analysis (NTA), also are limited in their ability to measure nanoparticles, due to the particles scattering less light than the limits of detection.
  • NTA nanoparticle tracking analysis
  • particles can diffuse in and out of the probe volume during NTA measurements, resulting in overcounting of smaller particles and under-counting of larger particles.
  • Improved optical methods are needed for the detection of particles, including nanoparticles, among which are EVs, which often are 500 nm or less in diameter.
  • a method of analyzing particles in a sample that includes: (a) contacting a sample comprising the particles with one or more optically detectable labels, thereby forming a staining solution, where: (i) the one or more optically detectable labels include a surface area probe or volume probe, where the surface area probe interacts with the particles stoichiometrically with respect to particle surface area or the volume probe interacts with the particles
  • the one or more optically detectable labels include a molecular marker-specific probe, where the molecular marker-specific probe interacts with a molecular marker of the particle stoichiometrically with respect to the number of molecules of the molecular marker that are associated with the particle, thereby forming particles that include particle-associated molecular marker-specific probe, where the optical signal from the particle-associated molecular marker-specific probe is proportional to the number of molecules of molecular marker associated with the particle; and (b) without physical separation or isolation of the particles, detecting the optical signal of the one or more particle-associated optically detectable labels generated in (i) and/or (ii), thereby analyzing the particles in the sample.
  • Also provided in certain aspects is a method of detecting, identifying, quantifying and/or determining the size of at least a first particle species in a sample that includes at two distinct particle species by: (a) contacting a sample containing at least two distinct particle species, where the distinct particle species differ from one another by size and/or by least one molecular marker associated with each particle species, with one or more optically detectable labels comprising a surface area probe or volume probe, where the surface area probe or volume probe interacts with at least a first particle species stoichiometrically with respect to particle surface area or volume, respectively, thereby forming particles that include particle-associated surface area probe or volume probe, where the optical signal from the particle- associated surface area probe or volume probe is proportional to the surface area or volume, respectively, of the first particle species; and/or (b) contacting the sample with one or more optically detectable labels that include a molecular marker-specific probe, where the molecular marker-specific probe interacts with a molecular marker of at least the first particle species
  • bound or “containing,” e.g. , “Npid-containing particle” can refer to a variety of different types of contact between, for example, a particle and its components (lipids, proteins, nucleic acids, carbohydrates, glycoproteins, glycolipids, phospholipids, phosphosphingolipids, etc.) or between a particle and an optically detectable label that can include, but is not limited to, covalent bonds or non-covalent interactions, non-limiting examples of which include van der Waals interactions, hydrogen bonding, ionic interactions, electrostatic interactions and/or hydrophilic or hydrophobic interactions.
  • the molecule that is the probe is also an optically detectable label, e.g. , di-8-ANEPPS.
  • the terms "associated,” “associated with” or “interact,” as used herein, also can refer to intercalation of the optically detectable label into the membrane, or binding of the optically detectable label to a molecular marker within or at the surface of the membrane vesicles, liposomes, extracellular vesicles and other lipid bilayer or lipid membrane containing particles.
  • the term “free” or “unbound,” as used herein, refers to molecules, including optically detectable labels, which are not in contact with the particle. "Free” or “unbound” optically detectable label, e.g.
  • the staining solution in the staining solution, generally is detected as a background signal or no signal, relative to the higher signal intensity of the optically detectable label when it is associated with a particle (i.e. , a particle-associated surface area probe or volume probe, or a particle-associated molecular marker-specific probe).
  • a particle i.e. , a particle-associated surface area probe or volume probe, or a particle-associated molecular marker-specific probe.
  • the particle is a nanoparticle of less than 1 micron in diameter.
  • the nanoparticles in the sample include at least one particle with a size of about 500 nm or less in diameter, between about 10 nm to about 200 nm in diameter, between about 50 nm to about 200 nm in diameter, between about 50 nm to about 150 nm in diameter, between about 10 nm to about 500 nm in diameter, between about 50 nm to about 200 nm in diameter, or between about 50 nm to about 150 nm in diameter.
  • the concentration of the particles in the sample is adjusted so the particle is optimally stained with, or associated with, or bound to, the optically detectable label.
  • the particle concentration can be adjusted to between about 1 x 10 3 particles ⁇ L to about 1 x 10 15 particles ⁇ L; between about 1 x 10 4 to about 1 x 10 13 to about 1 x 10 12 particles ⁇ L; between about 1 x 10 6 particles ⁇ L to about 1 x 10 12 particles ⁇ L;
  • the concentration of the particles in the sample is adjusted using a suitable buffer, such as an isotonic buffer, whereby the resulting staining solution contains a buffer.
  • the staining solution includes a surfactant, or a mixture of surfactants.
  • the surfactant could, in some embodiments, facilitate staining of the particles in the staining solution, such as the lipid bilayers of membrane vesicles, liposomes or extracellular vesicles.
  • the surfactant can be added to the staining solution in an amount of between about 0.001 % to about 0.5%; between about 0.002% to about 0.4% ;
  • the surfactant can be a nonionic poloxamer, such as the Synperonics, Pluronics and Kolliphor classes of poloxamers.
  • the surfactant can be a Pluronic poloxamer.
  • the Pluronic poloxamer can be Pluronic- 127.
  • analyzing the particles in the sample can include detecting the particles in the sample.
  • analyzing the particles refers to the detection and analysis of individual particles in the sample, such as by flow cytometry.
  • analyzing the particles "in bulk” means that the particles are analyzed as a whole, without resolution of the individual particles from one another, such as, for example, measuring the absorbance of a suspension of particles in a cuvette using a fluorimeter.
  • a bulk analysis can be distinguished, for example, from the detection and analysis of individual particles, such as by flow cytometry.
  • a bulk analysis also can include the detection and analysis of individual particles without distinguishing the individual particles from one another, such as identifying EVs in a sample without distinguishing them according to the cells from which they are derived and/or signature markers associated with different EVs.
  • analyzing the particles in the sample can include determining the surface area or volume of the particle based on the detected optical signal of the particle-associated surface area probe or volume probe, respectively.
  • the size of the particle can be determined based on the surface area or volume.
  • determining the size of the particle includes determining the diameter of the particle.
  • analyzing the particles in the sample can include determining the type and/or number of molecular markers associated with the particle based on the detected optical signal of the molecular marker-associated probe.
  • the particle can be identified and/or quantified based on the type and/or number of molecular markers associated with the particle.
  • the surface area probe or volume probe is a fluorescent label.
  • the molecular marker-specific probe is a fluorescent label.
  • any fluorescent label can be used in the methods provided herein including, but not limited to, a fluorophore, a tandem conjugate between more than one fluorophore, a fluorescent polymer, a fluorescent protein, or a fluorophore conjugated to a molecule that interacts with one or more particles of the sample.
  • the molecule that interacts with one or more particles of the sample includes, but is not limited to, a protein, an antibody, a lectin, a peptide, a nucleic acid, a carbohydrate or a glycan.
  • the molecule can interact with the particle in a manner that is proportional to the surface area or volume of the particle, or can bind or otherwise associate specifically with one or more molecular markers on the particle.
  • the molecule that interacts with one or more particles of the sample is an antibody, or a molecular marker- binding/associating fragment thereof.
  • Antibodies bind to specific antigens and contain two identical heavy chains and two identical light chains covalently linked by disulfide bonds. Both the heavy and light chains contain variable regions, which bind the antigen, and constant (C) regions.
  • one domain (V) has a variable amino acid sequence depending on the antibody specificity of the molecule.
  • the other domain (C) has a rather constant sequence common among molecules of the same class. The domains are numbered in sequence from the amino-terminal end.
  • the IgG light chain includes two immunoglobulin domains linked from N- to C-terminus in the order V L -C L , referring to the light chain variable domain and the light chain constant domain, respectively.
  • the IgG heavy chain includes four immunoglobulin domains linked from the N- to C- terminus in the order V H -C H 1-C H 2- C H 3, referring to the variable heavy domain, contain heavy domain 1 , constant heavy domain 2, and constant heavy domain 3.
  • the resulting antibody molecule is a four chain molecule where each heavy chain is linked to a light chain by a disulfide bond, and the two heavy chains are linked to each other by disulfide bonds. Linkage of the heavy chains is mediated by a flexible region of the heavy chain, known as the hinge region.
  • Fragments of antibody molecules can be generated, such as for example, by enzymatic cleavage. For example, upon protease cleavage by papain, a dimer of the heavy chain constant regions, the Fc domain, is cleaved from the two Fab regions (i.e. the portions containing the variable regions).
  • the IgA and IgG classes contain the subclasses lgA1 , lgA2, lgG1 , lgG2, lgG3, and lgG4. Any such antibody that is full length or a portion thereof that is less than full length, e.g.
  • the portion of an antibody can be a single chain variable fragment (scFv) of an antibody.
  • the antibody is a camelid single domain antibody.
  • the antibody or portion thereof is conjugated to a fluorophore.
  • the antibody is selected from among anti- CD61 , anti-CD171 , anti-CD325, anti-CD130, anti-GLAST, anti-EGFRvlll, anti-EGFR, anti-CD133, anti-CD15, anti-CD63, anti-CD9, anti-CD41 , anti-CD235, anti-CD54, anti-CD45 and anti-lgG.
  • the fluorophore is selected from among Dyl_ight488, a Brilliant Violet dye (exemplary of which are BV-421 , BV-510, BV-605 and the like), Pacific Blue, Chrome Orange, Brilliant Blue 515, PE, FITC, PE-Cy5.5, PE-Cy7, APC, Alexa647, APC-Alexa700 and APC-Alexa750.
  • Dyl_ight488, a Brilliant Violet dye exemplary of which are BV-421 , BV-510, BV-605 and the like
  • Pacific Blue Chrome Orange
  • Brilliant Blue 515 PE
  • PE-FITC PE-Cy5.5
  • PE-Cy7 PE-Cy7
  • APC Alexa647
  • At least one particle of the sample includes a lipid bilayer.
  • the particle containing a lipid bilayer can be a membrane vesicle, a lipoprotein, a liposome or an extracellular vesicle.
  • optically detectable labels associated with the particles in the samples analyzed according to the methods provided herein can be detected using a number of methods including, but not limited to, visual inspection, microscopy, spectroscopy, fluorescence spectroscopy, fluorescence imaging, imaging flow cytometry or flow cytometry.
  • the detection is by flow cytometry and the samples are analyzed by flow cytometry.
  • the optically detectable labels used in the analysis by flow cytometry are fluorescent labels.
  • one or more of the particles analyzed according to the methods provided herein includes membrane vesicles, lipoproteins, liposomes, extracellular vesicles or other particles containing a lipid bilayer membrane, or combinations thereof.
  • the surface area probe or volume probe that interacts with the particle containing a lipid bilayer membrane is selected from among di-8-ANEPPS, di-4-ANEPPS, F2N12S, FM-143, Cell Mask Orange, Cell Mask Green, Cell Mask Deep Red, a carbocyanine dye or a PKH dye.
  • the surface area probe or volume probe intercalates into the bilayer membrane.
  • the surface area probe is di-8-ANEPPS.
  • the surface area probe or volume probe is added in an amount such that the ratio of the amount surface area probe or volume probe (P) relative to the amount of lipid (L) in the particle, P/L, is adjusted whereby the surface area probe or volume probe interacts with the particles stoichiometrically with respect to particle surface area or volume, respectively.
  • the P/L ratio is between about 0.1 to about 0.25.
  • the molecular marker-specific probe is a fluorophore conjugated to a protein.
  • the protein is selected from among annexin V, cholera toxin B-subunit, anti-CD61 , anti-CD171 , anti-CD325, anti-CD130, anti-GLAST, anti-EGFRvlll, anti-EGFR, anti-CD133, anti- CD15, anti-CD63, anti-CD9, anti-CD41 , anti-CD235, anti-CD54 and anti-CD45.
  • the fluorophore conjugated to the protein conjugates is selected from among Dylight488, a Brilliant Violet dye, Pacific Blue, Chrome Orange, Brilliant Blue 515, PE, rhodamine, FITC, PE-Cy5.5, PE-Cy7, APC, Alexa647, APC-Alexa700 and APC-Alexa750.
  • physical separation or isolation of the particles includes filtration, washing the particles or precipitating the particles out of the sample or solution containing the particles. In some aspects, physical separation or isolation of the particles includes centrifugation or ultracentrifugation of the particles.
  • the flow cytometer has a configuration whereby light is collected from one side of the flow cell. In some aspects, the flow cytometer has a configuration whereby light is collected from both sides of the flow cell. In certain aspects, the detection range of the flow cytometer is between about 1 fluorescent molecule per particle to about 5, 10, 15, 20, 30, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500 or 2000 or more fluorescent molecules per particle. In some aspects, the resolution threshold of the flow cytometer is less than 200 fluorescent molecules per particle. In aspects, the resolution threshold of the flow cytometer is between about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 fluorescent molecules per particle to about 50, 100 or 150 fluorescent molecules per particle.
  • the particle is an extracellular vesicle, and, based on the detected optical signal of the molecular marker-specific probe, the type of molecular marker associated with the extracellular vesicle is determined. In some aspects, the cell and/or tissue of origin of the extracellular vesicle is identified based on the type of molecular marker associated with the extracellular vesicle.
  • one or more optical standard particles can be used to provide improved accuracy in determining the optical intensity of the optically detectable labels associated with the particles.
  • the optical standard particle can be a particle whose surface area or volume or diameter is predetermined by a method that does not use an optically detectable label, such as NTA, tunable resistive pulse sensing (TRPS) , electron microscopy (EM) or other methods.
  • the optical standard particle is capable of binding to or otherwise associating with an optically detectable label that is a surface area probe or a volume probe. The optical standard particle can then be contacted with an optically detectable label that is a surface area probe or volume probe and the intensity of the label associated with the optical standard particle obtained, thereby providing a correlation between surface area or volume, respectively, and optical intensity.
  • the optical standard particle is a particle containing molecular marker molecules that can be bound to or otherwise associated with one or more optically detectable labels that are molecular marker-specific probes.
  • the optical intensity of the molecular marker-specific probe-associated optical standard particle can be standardized against the measured optical intensity of a known external standard, thereby providing a correlation between optical intensity and the number of molecules of molecular marker associated with a particle.
  • the optical standard particle is a liposome or other lipid-containing particle. In aspects, the amount of lipid in the lipid-containing optical standard particle is known. In some aspects, the optical standard particle is a silica particle. In aspects, the silica particle includes a lipid bilayer. In some aspects, the optical standard particle is a bead. In some aspects, the bead can capture ligands that can bind to one or more molecular markers associated with a particle. In aspects, the ligand is an antibody. In certain aspects, the ligand is conjugated to an optically detectable label.
  • the optical standard particle is in a collection or preparation of optical standard particles that include a size distribution of optical reference particles, whereby a regression correlation between a distribution of sizes / surface area and optical intensities of the optical standard particles associated with an optically detectable label can be obtained.
  • the optical standard particle is in a collection or preparation of optical standard particles that include a distribution of numbers of molecular markers associated with each particle in the preparation, whereby a regression correlation between a distribution of numbers of molecules of molecular marker per optical standard particle and the optical intensities of the optical standard particles associated with an optically detectable label can be obtained.
  • the optical standard particles can be used for the analysis of particles according to the methods provided herein. In some aspects, the analysis is by flow cytometry.
  • the optical standard particle is a liposome, or a silica particle that includes a lipid bilayer.
  • the optically detectable label associated with the liposome or the lipid bilayer of the silica particle is di-8-ANEPPS or fluorescently labeled (e.g. , with Dyl_ight488) annexin V.
  • the optical standard particle is a bead that can bind to or otherwise associate with a ligand.
  • the ligand is an antibody. Any antibody as known and as described herein with respect to any aspect of the methods provided herein can be used as a ligand.
  • the antibody is labeled with a fluorophore.
  • the antibody is selected from among anti-CD61 , anti-CD171 , anti-CD325, anti-CD130, anti-GLAST, anti-EGFRvlll , anti-EGFR, anti-CD133, anti-CD15, anti-CD63, anti-CD9, anti-CD41 , anti-CD235, anti-CD54, anti-CD45 and anti-lgG.
  • the fluorophore is selected from among Dyl_ight488, a Brilliant Violet dye, Pacific Blue, Chrome Orange, Brilliant Blue 515, PE, FITC, PE-Cy5.5, PE-Cy7, APC, Alexa647, APC-Alexa700 and APC-Alexa750.
  • the methods provided herein are for simultaneously analyzing a plurality of particles of different size and/or having different molecular markers.
  • the different molecular markers can simultaneously be detected according to the methods provided herein, using optically detectable labels that are distinct from one another for each of the different molecular markers.
  • a panel of optical standard particles each associated with a distinct molecular marker conjugated to a distinct optically detectable label whereby, based on the measured optical intensities of the panel of optical standard particles, the optical intensities of the corresponding optically detectable labels associated with the molecular markers of the particles are measured with improved accuracy (e.g.
  • the analysis is by flow cytometry.
  • the panel of optical standard particles includes fluorescent beads.
  • the panel of optical standard particles includes beads that can bind to or otherwise associate with ligands, which in turn can be labeled with a fluorophore.
  • the ligand is an antibody.
  • the antibody is selected from among anti-CD61 , anti-CD171 , anti-CD325, anti-CD130, anti-GLAST, anti-EGFRvlll , anti-EGFR, anti-CD133, anti-CD15, anti-CD63, anti-CD9, anti-CD41 , anti-CD235, anti-CD54, anti-CD45 and anti-lgG.
  • the fluorophore is selected from among Dyl_ight488, a Brilliant Violet dye, Pacific Blue, Chrome Orange, Brilliant Blue 515, PE, FITC, PE-Cy5.5, PE-Cy7, APC, Alexa647, APC-Alexa700 and APC-Alexa750.
  • the size of an optical standard particle for use in the methods provided herein can be between about 20 nm to about 1 , 2, 3, 4, 5, 10 or more microns. In some aspects, the size of the optical standard particle is between about 20 nm to about 5 microns, about 30 nm to about 3 microns, about 40 nm to about 2 microns, about 50 nm to about 1 micron, about 50 nm to about 500 nm, about 50 nm to about 450 nm, about 50 nm to about 400 nm, about 100 nm to about 450 nm, or about 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480,
  • an optical standard particle that is not associated with an optically detectable label can be used, thereby improving accuracy by correcting the background optical signal obtained from the particle alone.
  • the optical standard particle is a bead.
  • the bead is coated with, bound to, or otherwise associated with a molecule.
  • the molecule is a polymer.
  • the polymer is polyethylene glycol (PEG).
  • the polymer is a protein that does not associate with an optically detectable label.
  • the protein is BSA.
  • Figure 1 shows the detection of fluorescently labeled particles by flow cytometry using a fluorescent trigger ( Figures 1 A-C) or a side scatter trigger ( Figures 1 D-F).
  • Figure 2 shows a comparison of size distribution profiles of vesicles using fluorescence intensity histograms obtained by flow cytometry ( Figures 2 A-D) or using nanoparticle diameter population histograms obtained by nanoparticle tracking analysis (NTA) ( Figures 2 E-H).
  • Figure 3 is a graph depicting the relationship between fluorescence intensity of a fluorescent probe associated with vesicles, and vesicle surface area.
  • Figure 4 shows the measurement of extracellular vesicles (EVs) in rat plasma by NTA ( Figures 4 A-C) or fluorescence triggered flow cytometry ( Figures 5 D-F), with their diameter calibrated using synthetic liposomes as reference particles.
  • Figure 4G shows plasma nanoparticle (EV) concentrations as measured by NTA and flow cytometry for eight animals.
  • Figure 5 shows the measurement of surface molecular markers of EVs in plasma from control plasma ( Figures 5 A, B) or ionophore-treated platelet rich plasma ( Figures 5 C, D) stained with di-8-ANEPPS and Dyl_ight488-Annexin V or Dylight488- anti-CD61 .
  • Figure 6 depicts fluorescence staining of polystyrene antibody capture beads coated with anti-lambda IgG and stained with a DyLight 488 conjugated antibody.
  • Figure 7 depicts specific fluorescence staining of synthetic liposomes containing phosphatidylserine (PS) in a population containing a mixture of synthetic liposomes that contain or do not contain PS.
  • PS phosphatidylserine
  • Figure 8 shows fluorescence staining of silica spheres coated with a lipid bilayer.
  • Figure 9 shows measurement of a fluorescence spectral shift to measure saturation of a lipid-containing particle using a membrane dye.
  • Figures 9 A-E depict measurements performed on synthetic liposomes having known amounts of associated lipid, and
  • Figure 9F depicts measurements performed on a sample of platelet-poor plasma (PPP).
  • PPP platelet-poor plasma
  • Figure 10 depicts measurement of light scatter in samples containing vesicles, using a fluorescence-based detection approach.
  • Figure 10A depicts buffer alone
  • Figure 10B depicts buffer + probe
  • Figure 10C depicts a sample preparation containing synthetic vesicles stained with probe
  • Figure 10D depicts the sample preparation of Figure 10C with added detergent (Triton X- 100; TX100)
  • Figure 10E depicts a platelet-free plasma preparation stained with dye
  • Figure 10F depicts the sample preparation of Figure 10E with added detergent (Triton X- 100; TX100).
  • Figure 1 1 depicts the analysis of EVs in human plasma using multiple markers.
  • Figure 1 1 A depicts measurements performed on synthetic liposomes
  • Figure 1 1 B depicts measurements performed on synthetic liposomes to which detergent is added (Triton X- 100; TX100)
  • Figure 1 1 C depicts measurements performed on platelet-rich plasma (PRP) supernatant
  • Figure 1 1 D depicts measurements performed on platelet-rich plasma (PRP) supernatant to which detergent is added (Triton X- 100; TX100).
  • optical methods for analyzing particles or vesicles with improved efficiency and accuracy can be used to analyze particles or vesicles of size ranging from about 1 nm in diameter to 100 microns ( ⁇ ) or more in diameter.
  • the analysis can include, but is not limited to, detection, quantitation, sizing and characterization of the particles, which can include determining the identity, i.e. , molecular content and origin of the particles (e.g. , cell/tissue of origin of an extracellular vesicle) .
  • the improved efficiency and accuracy of the methods provided herein permits the analysis of a wider range of particle sizes, including nanoparticles or nanovesicles of about 200 nm or less in diameter.
  • Samples containing particles of interest including microparticles, nanoparticles, liposomes, vesicles (unilamellar, multilamellar, e.g.) , lipoproteins, endosomes, viruses, viral particles, virus-like particles, apoptotic bodies and/or extracellular vesicles (EVs), are either at a particle concentration or are diluted to a sample particle concentration that facilitates optimal staining with an optically detectable label, detection of the label and analysis.
  • the samples are at, or can be diluted to, a final particle concentration of about 1 x 1 0 8 to about 1 x 10 10 particles / ⁇ .
  • the optimal dilution can, in embodiments, be determined by serial dilution of the sample in the presence of a constant amount of optically detectable label, thereby determining the optimal dilution (particle concentration) for enhanced signal from the label associated with the particles and low to negligible background signal from the unbound or free label.
  • the particle concentration of the diluted sample that produces optimal enhanced signal relative to background noise can independently be measured by a technique not involving contact with the optically detectable label, such as nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) or resistive pulse spectroscopy (RPS) , to determine the equivalent particle concentration of the optimally diluted sample.
  • NTA nanoparticle tracking analysis
  • TEM transmission electron microscopy
  • RPS resistive pulse spectroscopy
  • the dilution factor for analyzing membrane vesicles (e.g. , EVs) in the plasma is high due to the presence of high concentrations of proteins that can non-specifically compete with the vesicles for binding/association of the optically detectable label.
  • the dilution factor can be of the order of between 100-fold to 200-fold, or even 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000-fold or more.
  • the dilution factor for the analysis of membrane vesicles in the fluid can be lower, of the order of, for example, 20-fold, or in the range of between 5, 10, 15, 20, 25, 30, 25, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100-fold or higher.
  • the dilution factor can be anywhere from about 2-fold to about 5, 10, 15, 20, 25, 30, 25, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000-fold or higher. In certain embodiments, no dilution of the sample may be needed.
  • the sample can be treated to remove, in whole or in part, matter other than the particles such as undesired large particulates, cells, cellular debirs or other undissolved subject matter that does not include the particles.
  • the sample can be subjected to centrifugation at 2500g for one, two, three or more times, each step of centrifugation being performed for about 1 minute to about 20 minutes or more, for about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more minutes.
  • the centrifugation is performed two times for about 10 minutes each at 2500g.
  • the centrifugation is performed prior to dilution for optimal staining with an optically detectable label.
  • the sample containing an adjusted particle concentration as described above can be stained using one or more optically detectable labels.
  • the optically detectable labels and/or staining conditions are selected such that binding or other association of the labels to the particles is stoichiometric and/or saturable with respect to one or more of: (a) the surface area or volume of the particle; or (b) one or more specific molecular markers to which the optically detectable labels are bound, whereby the optical signal from the label is proportional to the surface area / volume and/or number of molecular markers of the particle, thereby providing information about the size, features and/or origin of the particle.
  • the staining can be performed before, after or contemporaneously with the sample dilution.
  • the optically detectable label can, in some embodiments, be a probe that can intercalate into the particle stoichiometrically with respect to the surface area and/or volume of the particle, thereby producing an optical signal that is proportional to the surface area and/or volume of the particle.
  • particles that are lipid vesicles e.g.
  • the fluorescent label di-8-ANEPPS (4-[2-[6-(dioctylamino)-2- naphthalenyl]ethenyl]1 -(3-sulfopropyl)-pyridinium) can bind to lipid membranes in a stoichiometric manner that is proportional to the surface area or volume of the liposomes.
  • An exemplary volume probe for use in any of the methods herein is carboxyfluorescein succinimidyl ester (CFSE).
  • the optically detectable label specifically binds or otherwise associates stoichiometrically with respect to one or more molecular components / markers of the particle (molecular marker-specific probe), thereby providing an optical signal that is specific for the marker and permits identification of the type of particle based on the type of detected marker.
  • the molecular marker-specific probe binds or otherwise associates with the molecular marker in a stoichiometric manner proportional to the number of molecules of molecular marker per particle.
  • a "molecular marker” is a molecule that is a specific component or ligand of a particular type of particle.
  • the molecular marker can be present anywhere in the interior or on the surface of the particle, or can be associated with the membrane when particle is a vesicle (e.g. , membrane vesicles, liposomes, EVs), and detection of the molecular marker can identify the type of particle associated with the molecular marker.
  • the molecular marker can be present on the surface of the particle.
  • annexin V has a specific binding affinity for phosphatidyl serine (PS), which is a surface molecular marker of many cell-derived EVs, membrane vesicles and liposomes.
  • the number of cell-derived EVs or other PS-containing vesicles can be determined by staining with an optically labeled annexin V, e.g. , annexin V conjugated to the fluorescent label, Dylight488- succinimidyl ester.
  • platelet-derived extracellular vesicles have CD61 as a molecular marker, which can be detected using anti-CD61 that has been labeled with an optically detectable label. Identifying the type of particle can include identifying its origin or source. For example, when the particle is an EV, as indicated in the aforementioned example, the detection of CD61 in the EV can identify the EV as originating from platelets.
  • the particles can be stained with both a surface area probe or volume probe for optical detection, and a molecular marker-specific optical label.
  • concentration of the optically detectable label, the choice of staining buffer, the temperature during staining and/or the staining time can be adjusted to achieve stoichiometric incorporation of the optically detectable label in the interior and/or surface of the particle.
  • Any optically detectable label that shows enhanced intensity e.g., color, fluorescence, luminescence, bioluminescence, chemiluminescence and light scatter
  • any optically detectable label that shows enhanced intensity e.g., color, fluorescence, luminescence, bioluminescence, chemiluminescence and light scatter
  • stoichiometric refers to the association or binding of a surface area/volume probe or molecular marker-specific probe to a particle in a manner that is proportional to the surface area/volume of a particle (for a surface area/volume probe) or the number or concentration of molecules of molecular marker-specific probe associated with the particle, whereby, based on the intensity of the signal generated from the optically detectable label associated with the probe, the surface area of the particle or the number/concentration of molecules of marker- specific probe, respectively, can be determined.
  • saturated refers to the amount of probe (surface area or molecular-marker specific) which, when associated with or bound to a particle, generates a signal from an optically detectable label associated with the probe that does not substantially increase when more probe is added to the sample containing the particle.
  • probe surface area or molecular-marker specific
  • adding further amounts of the probe does not substantially increase the signal generated by the optically detectable label by more than 0% 0.05%, 0.1 % , 0.2%, 0.3%, 0.4%, 0.5% , 0.6%, 0.75. 0.8%.
  • stoichiometric binding/association of the surface probe or molecular marker-specific probe to a particle is achieved when the
  • the binding/association of the probe to/with the particle is in an amount that is saturating for the signal intensity of the optically detectable label associated with the probe.
  • the optically detectable label used in the methods is saturable when bound/associated as a probe to a particle.
  • di-8-ANEPPS is a saturable optically detectable surface area probe.
  • lipid-containing particles such as liposomes, EVs or other lipid-containing vesicles
  • the probe when probe is added to such particles, the probe often becomes associated with the particles by intercalation into the lipid bilayer membranes.
  • the optimal probe to lipid ratio is considered as "approaching saturation” rather than becoming saturated because saturation can lead to self-quenching among the large number of intercalated probe molecules.
  • a probe amount “approaching saturation” or that “approaches saturation” can be used interchangeably with “saturable,” “saturated,' “saturating” and the like and refers to the amount of probe (surface area or molecular-marker specific) which, when associated with or bound to a particle, generates a signal from an optically detectable label associated with the probe that does not substantially increase when more probe is added to the sample containing the particle.
  • the methods provided herein do not include a physical separation or isolation.
  • physical separation or isolation means that the non-particle reaction components of the staining reaction are substantially removed from the presence of the stained particles (e.g. , the container in which the stained particles are present) by methods such as filtration, precipitation, washing or centrifugation, including ultracentrifugation.
  • non-particle reaction components or “non-particle components” of the staining reactions refers to all components of the staining reaction other than the particles, some or all of which include bound (particle-associated) optically detectable label; the non-particle components can include buffers, salts, surfactants, unbound (free) optically detectable label and other components that are present in the staining reaction by which one or more optically detectable labels are incorporated into the particles.
  • Substantial removal of the non-particle components of the staining reaction means that at least about 50, 60, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or more percent of the non-particle components of the staining reaction, i.e. , substantially all components of the reaction other than the stained particles, are removed by one or more physical separations, e.g. , washing or centrifugation.
  • the resulting stained samples can be diluted by a factor sufficient to reduce the background signals associated with optically detectable labels that are not bound to or associated with the particles, without physical separation or isolation.
  • the analysis of the stained particles by dilution of the staining reaction mix can provide improved efficiency by reducing the number of steps used to process the particles prior to analysis.
  • the analysis of microparticles or nanoparticles generally involves handling small volumes of samples containing the particles and the repeated washing or centrifugation / ultracentrifugation of small volumes can lead to loss of a fraction of the particles, thereby reducing the accuracy of analysis.
  • the dilution can be by a factor sufficient to minimize interference from the signal associated with free optically detectable label that is not associated with the particle, while maintaining enhanced signal from the optically detectable label that is bound to or otherwise associated with the particle.
  • particle-associated label is analyzed in the presence of free label, the free label generating a minimal background signal that does not interfere with the detection of signal generated by the particle-associated optically detectable label.
  • the dilution also can be by a factor whereby multiple particles are not detected simultaneously, i.e.
  • the dilution can be by a factor of anywhere from about 2-fold to about 5, 10, 15, 20, 25, 30, 25, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000-fold or higher, depending on the sample.
  • no dilution of the staining reaction solution may be needed.
  • the dilution of a sample stained with 500 nM di-8-ANEPPS and 50 nM molecular marker-specific probe can be by a factor of about 1000.
  • the label-bound particles in the sample that is diluted, post-staining, as described above, can be detected, characterized (e.g. , its molecular components identified and/or its origin identified - e.g., if the particle is an EV, its cell/tissue of origin can be identified) and/or quantitated based on detection of the bound optically-detectable label.
  • Any label that can be detected by optical means can be used for analyses of the particles.
  • the sample can be analyzed by visual inspection or can be illuminated by an instrument capable of detecting an optically detectable label associated with a particle.
  • the illumination wavelength can be tailored to detection of a particular particle-associated optically detectable label, whether the label is a surface area probe or volume probe, or a molecular marker-specific probe.
  • the intensity of the signal from the particle-associated optically detectable label can be measured by the instrument detecting such signal or by a separate instrument capable of measuring the intensity of a signal from an optically detectable label associated with a particle.
  • Exemplary optical elements for selecting and dispersing light can include, but are not limited to, band pass filters, dichroic mirrors or optical gratings for filtering or dispersing light onto a detector.
  • Exemplary detectors can include, but are not limited to, a photomultiplier tube (PMT), an avalanche photodiode, an avalanche photodiode array, a silicon-PMT, a hybrid PMT, a photodiode array, a charged cathode device (CCD), an electron multiplied CCD, a CMOS sensor detector, or any suitable photodetector.
  • the measured intensity can be used to characterize the particles in the sample according to presence or absence of the particle, type / identity of the particle, source/origin of the particle, number of molecules of molecular marker on the particle, size of the particle or quantity of the particle.
  • Samples that contain particles for analysis according to the methods provided herein generally include particles in a liquid medium.
  • the particles can be analyzed by detection, identification, characterization according to the presence of one or more molecular markers associated with the particles, or characterization according to the size of the particles. Any samples containing particles in a liquid can be analyzed according to the methods provided herein.
  • any aqueous or organic liquid medium containing particles, where the particles are not dissolved in the liquid medium, are contemplated for use in the methods herein.
  • the liquid medium can be a solution that includes solutes dissolved in the liquid medium, such as buffers.
  • the samples include a suspension of particles, or a colloidal suspension of particles, in the liquid medium.
  • Exemplary liquid media containing particles that can be analyzed according to the methods provided herein include, but are not limited to, blood, milk, water, solutions containing particles such as membrane vesicles, lipoproteins, viruses, virus-like particles, apoptotic bodies, synthetic liposomes or extracellular vesicles, and biological fluids other than blood such as plasma, serum, urine, saliva, seminal fluid, lavages (e.g.
  • bronchoalveolar gastric, peritoneal, ductal, ear, arthroscopic
  • cervical fluid cervicovaginal fluid, cerebrospinal fluid, vaginal fluid, breast fluid, breast milk, synovial fluid, semen, seminal fluid, sputum, cerebral spinal fluid, tears, mucus, interstitial fluid, follicular fluid, amniotic fluid, aqueous humor, vitreous humor, peritoneal fluid, ascites, sweat, lymphatic fluid, lung sputum or fractions or components thereof.
  • the sample containing particles is a biological sample.
  • the biological sample includes a biological fluid.
  • the biological fluid in the biological sample can include, but is not limited to, blood, plasma, serum, urine, saliva, seminal fluid, lavages (e.g.
  • the biological fluid is blood, plasma or serum. In some embodiments, the biological fluid is cerebrospinal fluid.
  • the biological sample is extracted from a cell or tissue sample of a subject, such as a biopsy sample (e.g. , cancer biopsy), or is extracted from normal or cancer cell samples or normal or cancer tissue samples where the cell or tissue samples can be derived, e.g. , from the liver, lung, kidney, spleen, pancreas, colon, skin, bladder, eye, brain, esophagus, head, neck, ovary, testes, prostate, the like or combination thereof.
  • the biological sample that is extracted from a cell or tissues sample of a subject includes a biological fluid.
  • the biological sample includes particles derived from a cancer biopsy, a cancer cell or a cancer tissue.
  • Cancer biopsy samples, cancer cell types or cancer tissue types from which particles can be present in the biological sample include, but are not limited to, liver cells (e.g. , hepatocytes), lung cells, spleen cells, pancreas cells, colon cells, skin cells, bladder cells, eye cells, brain cells, esophagus cells, cells of the head, cells of the neck, cells of the ovary, cells of the testes, prostate cells, placenta cells, epithelial cells, endothelial cells, adipocyte cells, kidney cells, heart cells, muscle cells, blood cells (e.g.
  • the cancer is a glioblastoma.
  • the cancer is ovarian, lung, bladder or prostrate cancer.
  • the biological sample that includes particles derived from a cancer biopsy, a cancer cell or a cancer tissue further includes a biological fluid.
  • the biological fluid is blood, plasma, serum, saliva, urine or cerebrospinal fluid.
  • the cancer is ovarian, lung, bladder or prostrate cancer and the biological fluid is saliva, urine or serum.
  • the cancer is brain cancer.
  • the cancer is brain cancer and the biological fluid is cerebrospinal fluid.
  • the brain cancer is glioblastoma.
  • a sample can be blood and sometimes a blood fraction (e.g. , plasma or serum).
  • blood encompasses whole blood or any fractions of blood, such as serum and plasma as conventionally defined, for example.
  • Blood also contains buffy coats. Buffy coats sometimes are isolated by utilizing a ficoll gradient. Buffy coats can comprise white blood cells (e.g. , leukocytes, T-cells, B-cells, platelets, and the like) and samples extracted from buffy coats can include particles, e.g. , extracellular vesicles (EVs), derived from these cells.
  • Blood plasma refers to the fraction of whole blood resulting from centrifugation of blood treated with anticoagulants.
  • Blood serum refers to the watery portion of fluid remaining after a blood sample has coagulated. Fluid or tissue samples often are collected in accordance with standard protocols hospitals or clinics generally follow. For blood, an appropriate amount of peripheral blood (e.g. , between 3-40 milliliters) often is collected and can be stored according to standard procedures prior to or after preparation.
  • a sample obtained from a subject can contain cellular elements or cellular remnants.
  • cancer cells may be included in the sample.
  • the sample is obtained from a human subject.
  • the human subject is a cancer patient and in some embodiments, the human subject does not have cancer.
  • any particles that can bind to or otherwise associate with an optically detectable label are contemplated for analysis according to the methods provided herein.
  • the particles can occur in nature, or can be synthetic or artificially prepared.
  • the particles described herein and elsewhere in this application can be the particles of interest, i.e. , the particles that are desired to be analyzed by the methods, or they can be used as optical standard particles for improved accuracy of measurement of the optical intensity.
  • the particles can be inert particles that can associate with an optically detectable label or can be modified for association with an optically detectable label.
  • Such particles can include metalloids, non-limiting examples of which include boron and silicon, the like and combinations thereof.
  • a particle sometimes can include, consist essentially of, or consist of, silica (e.g. , silicon dioxide (i.e. , Si0 2 )).
  • a particle sometimes can include one or more metals including, but not limited to, iron, gold, copper, silver, platinum, aluminum, titanium, tantalum, vanadium, the like, oxides thereof and combinations thereof.
  • a particle sometimes can include glass (e.g. controlled-pore glass (CPG)), nylon, Sephadex®,
  • Sepharose® cellulose, a magnetic material or a plastic material.
  • a particle sometimes is a polymer or includes more than one polymer.
  • Non-limiting examples of polymers include polypropylene (PP), polyethylene (PE), polyamide, high-density polyethylene (HDPE), low-density polyethylene (LDPE), polyester,
  • PVDF polyvinylidenedifluoride
  • PET polyethylene teraphthalate
  • PVC polyvinyl chloride
  • PTFE polytetrafluoroethylene
  • PS polystyrene
  • high-density polystyrene acrylnitrile butadiene styrene copolymers, crosslinked polysiloxanes, polyurethanes, (meth)acrylate-based polymers, cellulose and cellulose derivatives, polycarbonates, ABS, tetrafluoroethylene polymers, poly(2-hydroxy ethyl methacrylate), poly(N-vinyl pyrrolidone), poly(methyl methacrylate), polyvinyl alcohol), poly(acrylic acid), polyacrylamide, poly(ethylene-co-vinyl acetate), poly(ethylene glycol),
  • PVDF polyvinylidenedifluoride
  • PET polyethylene teraphthalate
  • PVC polyvinyl chloride
  • PTFE polytetrafluoroethylene
  • the particles can be solid particles or particles that contain internal voids.
  • the particles can have a regular (e.g. , spheroid, ovoid) or irregular shape (e.g. , rough, jagged), and sometimes can be non-spherical (e.g. , angular, multi-sided).
  • the particles include membrane vesicles.
  • a membrane vesicle refers to a particle that includes fluid enclosed within a lipid-containing outer shell.
  • the enclosed fluid can include additional components, such as proteins and small molecules.
  • a lipid molecule typically includes at least one hydrophobic chain and at least one polar head. When exposed to an aqueous environment, lipids often will self assemble into structures that minimize the surface area exposed to a polar (e.g. , aqueous) medium.
  • Lipids sometimes assemble into structures having a single or monolayer of lipid enclosing a non-aqueous environment, and lipids sometimes assemble into structures comprising a bilayer enclosing an aqueous environment.
  • the polar portion of lipids e.g. , the head of the molecule in the case of phospholipids and other lipids commonly found in cell substrates
  • the non-polar portion of the lipid often is oriented towards the polar, aqueous environment, allowing the non-polar portion of the lipid to contact the non-polar environment.
  • a vesicle also can be a lipid bilayer configured as a spherical shell enclosing a small amount of water or aqueous solution and separating it from the water or aqueous solution outside the vesicle.
  • Membrane vesicles also can contain a fluid with, optionally, one or more molecular components, enclosed within a lipid bilayer.
  • vesicles Because of the fundamental similarity to a cell wall, vesicles have been used to study the properties of lipid bilayers. Vesicles also are readily manufactured. A sample of dehydrated lipid spontaneously forms vesicles, when exposed to water.
  • Spontaneously formed vesicles can be unilamelar (single-walled) or multilamellar (many-walled) and are of a wide range of sizes from tens of nanometers to several micrometers.
  • a lipid bilayer typically includes a sheet of lipids, generally two molecules thick, arranged so that the hydrophilic phosphate heads point towards a hydrophilic aqueous environment on either side of the bilayer and the hydrophobic tails point towards the hydrophobic core of the bilayer. This arrangement results in two “leaflets” that are each a single molecular layer. Lipids self-assemble into a bilayer structure due to the hydrophobic effect and are held together by non-covalent forces that do not involve formation of chemical bonds between individual molecules.
  • lipid bilayers are natural, and in certain embodiments lipid bilayers are artificially generated. Natural bilayers often are made mostly of phospholipids, which have a hydrophilic head and two hydrophobic tails (e.g. , lipid tails), and form a two-layered sheet as noted above, when exposed to water or an aqueous environment. The center of this bilayer contains almost no water and also excludes molecules like sugars or salts that dissolve in water, but not in oil. Lipid tails also can affect lipid composition properties, by determining the phase of the bilayers, for example. A bilayer sometimes adopts a solid gel phase state at lower temperatures and undergoes a phase transition to a fluid state at higher
  • Artificial bilayers of membrane vesicles can be any bilayers assembled through artificial means, as opposed to bilayers that occur naturally (e.g. , cell walls, lipid bilayers that cover various sub-cellular structures).
  • bilayers e.g. , viscosity or fluidity of lipid bilayers.
  • Phospholipids with certain head groups can alter the surface chemistry of a bilayer.
  • bilayer constituents that can alter the surface chemistry of bilayers include fats, lecithin, cholesterol, proteins, phospholipids (e.g. , phosphatidic acid (phosphatidate), phosphatidylethanolamine (e.g. , cephalin), phosphatidylcholine (e.g.
  • phosphatidylserine and phosphoinositides such as phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP), phosphatidylinositol bisphosphate (PIP2) and phosphatidylinositol triphosphate (PIP3), phosphatidylglycerol, ceramide
  • PI phosphatidylinositol
  • PIP phosphatidylinositol phosphate
  • PIP2 phosphatidylinositol bisphosphate
  • PIP3 phosphatidylinositol triphosphate
  • phosphorylglycerol phosphorylglycerol
  • phosphosphingolipids glycolipids including gangliosides, surfactants, the like and combinations thereof.
  • lipid compositions e.g. , monolayers and/or bilayers
  • lipid compositions can be found naturally or generated artificially.
  • compositions include monolayers (e.g. , micelles), supported lipid bilayers, linear lipid bilayers and the like.
  • a protein, glycoprotein, glycolipid, nucleic acid or carbohydrate often is inserted into a structure (e.g. , monolayer and/or bilayer) formed by the lipid or amphiphilic material composition, or is encapsulated within the interior of the structure (membrane vesicle or other particle as described herein).
  • a protein that is inserted into the structure can be water soluble, detergent-solubilized or incorporated into a lipid bilayer (e.g. , vesicle, liposome) or a lipid monolayer (e.g. , micelle) in some embodiments.
  • membrane vesicles can include lipoproteins, endosomes, apoptotic bodies, viruses, virus particles and virus-like particles.
  • Endosomes are membrane- bound vesicles, formed via a complex family of processes collectively known as endocytosis, and found in the cytoplasm of virtually every animal cell. The basic mechanism of endocytosis is the reverse of what occurs during exocytosis or cellular secretion or the release of extracellular vesicles (EVs, e.g. , ectosomes, exosomes) as it involves the invagination (folding inward) of a cell's plasma membrane to surround macromolecules or other matter diffusing through the extracellular fluid.
  • EVs extracellular vesicles
  • the encircled foreign materials are then brought into the cell, and following a pinching-off of the membrane (termed budding), are released to the cytoplasm of the cell in a saclike vesicle.
  • the sizes of the endosomal vesicles can vary and generally are nanoparticles. Endosomes larger than 100 nanometers in diameter typically are referred to as vacuoles.
  • Viruses, virus particles and virus-like particles can include a lipid bilayer and, in embodiments, carry proteins on their surface, including envelope proteins, coat proteins and cellular membrane proteins. "Naked viruses” generally lack surface proteins and can be modified to include surface proteins (e.g., by insertion of the proteins into the outer lipid bilayer of the virus). Viruses include for example, but are not limited to, retroviruses and DNA viruses. Virus particles can include the fully or partially assembled capsid of a virus. A viral particle may or may not contain nucleic acid. Virus particles generally include one or more of or two or more of the following: genetic material made from either DNA or RNA; a protein coat that protects the genetic material; and in some embodiments an envelope of lipids that surrounds the protein coat when they are outside a cell.
  • Lipoproteins are globular, micelle-like particles that include a non-polar core of acylglycerols and cholesteryl esters surrounded by an amphiphilic coating of protein, phospholipid and cholesterol. Lipoproteins have been classified into five broad categories on the basis of their functional and physical properties: chylomicrons, which transport dietary lipids from intestine to tissues; very low density lipoproteins (VLDL); intermediate density lipoproteins (IDL); low density lipoproteins (LDL); all of which transport triacylglycerols and cholesterol from the liver to tissues; and high density lipoproteins (HDL) , which transport endogenous cholesterol from tissues to the liver. Lipoprotein particles undergo continuous metabolic processing and can have variable properties and compositions.
  • Lipoprotein densities can increase without decreasing particle diameter because the density of their outer coatings is less than that of the inner core.
  • the protein components of lipoproteins are known as apolipoproteins. At least nine apolipoproteins are distributed in significant amounts among the various human lipoproteins.
  • Apoptotic bodies are released during apoptosis (programmed cell death).
  • apoptosis programmed cell death
  • the breakdown components are packaged into apoptotic bodies, which can include membrane bound "sacs" that contain nucleic acids, proteins and lipids.
  • apoptotic bodies When the ability of neighboring cells and/or macrophages to clear these breakdown components is overwhelmed by high numbers of apoptotic bodies ("excessive" apoptosis) or defects in clearing the bodies, apoptotic bodies are released into circulation and can be detected in blood plasma or serum (Holdenrieder et al, 2001 a; Holdenrieder et al, 2001 b; Holdenrieder et al, 2001 c; Lichtenstein et al, 2001 ) . Above-average levels of apoptotic bodies in the bloodstream have been correlated, e.g. , with the presence tumors and cancers.
  • apoptotic body can contain nucleic acids, proteins, lipids, but no nucleus, although it may contain fragmented nuclei.
  • apoptotic bodies are less than 10 microns in size, generally between about 25 nm, 50nm, 75 nm or 100 nm to about 150 nm, 200 nm, 250, 300 nm, 350 nm, 400 nm, 400 nm, 500 nm, 1 micron, 1 .5 micron, 2 microns, 2.5 microns, 3 microns, 3.5 microns, 4 microns, 4.5 microns or 5 microns in size.
  • a liposome is an artificially prepared vesicle that includes at least one lipid bilayer and also can be made of naturally occurring or synthetic lipids, including
  • Liposomes can include MLV (multilamellar vesicles) , SUV (Small Unilamellar Vesicles) , LUV (Large Unilamellar Vesicles) and GUV (Giant Unilamellar Vesicles).
  • Unilamellar vesicles generally contain a single lipid bilayer, while multilamellar vesicles generally include more than one lipid bilayer.
  • multivesicular liposome refers to man-made, microscopic lipid vesicles containing lipid membranes enclosing multiple concentric or non-concentric aqueous chambers.
  • lipids can be used to make liposomes, including neutral lipids and amphipathic lipids.
  • neutral lipids include diglycerides, such as diolefin, dipalmitolein; propylene glycol esters such as mixed diesters of caprylic/capric acids on propylene glycol; triglycerides such as triolein, tripalmitolein, trilinolein, tricaprylin and trilaurin; vegetable oils, such as soybean oil; lard or beef fat; squalene;
  • amphipathic lipids include those with net negative charge, zero net charge, and net positive charge at pH 7.4. These include zwitterionic, acidic or cationic lipids.
  • amphipathic lipids include, but are not limited to, phosphatidylglycerol (PG), cardiolipin (CL), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol,
  • PC phosphatidylcholine
  • PE phosphatidylethanolamine
  • DITAP diacyl trimethylammonium propane
  • DOPC or DC18: 1 PC 1 ,2-dioleoyl-sn-glycero- 3-phosphocholine
  • DLPC or DC12:0PC 1 ,2-dilauroyl-sn-glycero-3-phosphocholine
  • DMPC or DC14:0PC 1 ,2-dimyristoyl-sn-glycero-3-phosphocholine
  • DPPC or DC16:0PC 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine
  • DSPC or DC18:0PC 1 ,2- distearoyl-sn-glycero-3-phosphocholine
  • DAPC or DC20:0PC 1 ,2diarachidoyl-sn- glycero-3-phosphocholine
  • DBPC or DC20:0PC 1 ,2
  • DPPG 1 ,2-dipalmitoyl-sn-glycero-3-phosphoglycerol
  • DOPG 1 ,2- dioleoyl-sn-glycero-3-phosphoglycerol and combinations thereof.
  • lipoproteins, gangliosides, cholesterol or plant sterols can be used to make, or are a part of, liposomes.
  • lipid-polymer conjugates and liposomes are disclosed in U.S. Patent No. 5,631 .018, which is incorporated herein by reference in its entirety.
  • the particles can be extracellular vesicles (EVs).
  • extracellular vesicles can include membrane vesicles secreted from cell surfaces (ectosomes), internal stores (exosomes), cancer cells (oncosomes), or released as a result of apoptosis and cell death.
  • EVs can include additional components such as lipoproteins, proteins, nucleic acids, phospholipids, amphipathic lipids, gangliosides and other particles contained within the lipid membrane or encapsulated by the EVs.
  • Non-limiting examples of normal or cancer cell types that can release EVs include liver cells (e.g. , hepatocytes) , lung cells, spleen cells, pancreas cells, colon cells, skin cells, bladder cells, eye cells, brain cells, esophagus cells, cells of the head, cells of the neck, cells of the ovary, cells of the testes, prostate cells, placenta cells, epithelial cells, endothelial cells, adipocyte cells, kidney cells, heart cells, muscle cells, blood cells (e.g. , white blood cells, platelets), the like and combinations of the foregoing.
  • EVs are involved in cell-cell communication, their characterization casts light upon their role in normal physiology and pathology. EVs in biological fluids fluids including saliva, urine and sera are being interrogated as biomarkers of ovarian, lung, bladder and prostate cancers.
  • Glioblastoma is the most common form of primary brain cancer and is one of the deadliest of human cancers. Glioblastoma cells release extracellular vesicles (EVs) containing amplified and mutated genetic materials derived from the tumor. The circulating EVs significantly exceed tumor-derived circulating tumor cells and tumor derived circulating DNA and RNA. The released EVs appear in the local vesicles (EVs) containing amplified and mutated genetic materials derived from the tumor. The circulating EVs significantly exceed tumor-derived circulating tumor cells and tumor derived circulating DNA and RNA. The released EVs appear in the local vesicles (EVs) containing amplified and mutated genetic materials derived from the tumor. The circulating EVs significantly exceed tumor-derived circulating tumor cells and tumor derived circulating DNA and RNA. The released EVs appear in the local vesicles (EVs) containing amplified and mutated genetic materials derived from the tumor. The circulating
  • GBM cerebrospinal fluid
  • EVs are abundant in various biological fluids, including blood, urine, and
  • EVs in biofluids can be heterogeneous. While multispectral optical methods can detect vesicles that have different molecular markers, the small average size of EVs can result in small optical signals from labels bound to or otherwise associated with these small particles, making it a challenge to analyze the EVs by optical methods.
  • EVs can be released by all normal and cancer cells. With a mean diameter of ⁇ 100- 200 nm, however, individual EVs have ⁇ 1 /10,000 the surface area and ⁇ 1 /1 ,000,000 the volume of a whole cell, making them difficult to detect using available single cell analysis tools, including conventional flow cytometry. As a result, most proteomic and genomic analysis is performed in bulk on thousands or millions of EVs. However, EVs in biofluids come from many different cell types, and from different locations from within the cell (exosomes secreted from intracellular multi-vesicular bodies, ectosomes/microvesicles shed from the plasma membrane surface, membrane fragments released as a result of cell apoptosis, necrosis, etc).
  • the signature from tumor EVs may be lost in the background of vesicles from other sources.
  • Single EV measurement approaches such as nanoparticle tracking analysis (NTA) and resistive pulse sensing (RPS) can report particle concentrations, but provide no information on the cell of origin.
  • NTA nanoparticle tracking analysis
  • RPS resistive pulse sensing
  • the size of the particles (e.g. , inert particles, liposomes or EVs) analyzed according to the methods provided herein can include particles in the size range (average length, width or diameter) of about or at 10 nm to about or at 5 microns, but generally are in the range of about or at 50 nm to about or at 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900 or 950 nm or 1 .0 to 1 .5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0 microns.
  • optically detectable labels can include, for example, chromophores, chemiluminescent moieties, bioluminescent moieties, fluorescent moieties and metals.
  • Such labels can be detected, for example, by visual inspection, by spectroscopy, by fluorescence spectroscopy, by fluorescence imaging (e.g. , using a fluorescent microscope or fluorescence stereomicroscope), by flow cytometry and the like.
  • Exemplary chromophores include, but are not limited to, 3,3'-diaminobenzidine (DAB); 3-amino-9-ethyl carbazole (AEC); Fast Red; FD&C Yellow 5 (Tartrazine); Malachite Green Carbinol hydrochloride; Crocein Scarlet 7B (Dark Red); Erioglaucine (Dark Blue); Crystal Violet (Dark Purple); Bromophenol Blue; Cobalt(ll) Chloride Hexahydrate (Red); Basic Violet 3; Acid Blue 9; Acid Red 71 ; FD&C Blue 1 (Brilliant Blue FCF); FD&C Red 3 (Erythrozine); and FD&C Red 40 (Allura Red AC).
  • DAB 3,3'-diaminobenzidine
  • AEC 3-amino-9-ethyl carbazole
  • Fast Red FD&C Yellow 5
  • Malachite Green Carbinol hydrochloride Crocein Scarlet 7B (Dark Red); Er
  • fluorophores include, but are not limited to, di-8-ANEPPS, di-4-ANEPPS, a carbocyanine dye (e.g. , DiO, DiL), a PKH dye (exemplary of which are PKH-26 and PKH-67), Dylight488, Brilliant Violet, Pacific Blue, Chrome Orange, Brilliant Blue 515, phycoerythrin (PE), rhodamine, fluorescein, FITC, PE-Cy5.5, PE-Cy7, APC,
  • the fluorescent reagent can be chosen based on desired excitation and emission spectra.
  • fluorescent reagents are macromolecules that emit an optically detectable signal, including fluorescent proteins, such as a green fluorescent protein (GFP) or a red fluorescent protein (RFP).
  • fluorescent proteins such as a green fluorescent protein (GFP) or a red fluorescent protein (RFP).
  • GFP green fluorescent protein
  • RFP red fluorescent protein
  • DNA sequences encoding proteins that can emit a detectable signal or that can catalyze a detectable reaction, such as luminescent or fluorescent proteins are known and can be used in the methods provided herein.
  • Exemplary genes encoding light-emitting proteins include, for example, genes from bacterial luciferase from Vibrio harveyi (Belas et al. , (1982) Science 218:791 -793), bacterial luciferase from Vibrio fischerii (Foran and Brown, (1988) Nucleic acids Res.
  • the luxA and luxB genes of bacterial luciferase can be fused to produce the fusion gene (Fab 2 ) , which can be expressed to produce a fully functional luciferase protein (Escher et al. , (1989) PNAS 86: 6528-6532) .
  • the optically detectable label can be conjugated to a molecule (e.g. , a protein, an antibody, a lectin, a peptide, a nucleic acid, a carbohydrate, a glycan and the like) that binds to or otherwise associates with a molecular marker on the particle, or associates with / intercalates into the particle membrane (e.g. , when the particle is a vesicle, liposome or EV) .
  • a molecule e.g. , a protein, an antibody, a lectin, a peptide, a nucleic acid, a carbohydrate, a glycan and the like
  • the particles can be analyzed by Raman flow cytometry using Surface Enhanced Raman Scattering (SERS) from metal nanoparticles (Nolan et al. , Methods, 57:272-279 (2012).
  • SERS Surface Enhanced Raman Scattering
  • the particles are analyzed by flow cytometry.
  • Any conventional flow cytometer including a spectral flow cytometer, a hyperspectral flow cytometer, or an imaging flow cytometer can be used in the methods provided herein.
  • the flow cytometry analysis is fluorescence-based rather than light scatter-based. Without being bound by theory, it is believed that when the particles are nanoparticles, e.g.
  • Fluorescence-based flow cytometry can be useful, for example, in the analysis of nanoparticles.
  • extracellular vesicles EVs
  • EVs extracellular vesicles
  • the analysis of such nanoparticles by fluorescence based flow cytometry, according to the methods provided herein, can quantitatively determine the size of the nanoparticles by stoichiometric staining of the surface area using a fluorescent surface area probe or volume probe, whereby the fluorescent intensity is proportional to the surface area or volume, respectively.
  • fluorescence-based flow cytometry permits the sensitive detection of the number and type of molecular markers, such as surface antigens, on the nanoparticles, such as EVs, thereby providing information regarding the tissues / cells from which they originate, as well as whether the tissues / cells are cancerous (based on the number of EVs, number of molecules of molecular marker and/or type of molecular markers detected on the EVs).
  • molecular markers such as surface antigens
  • Exemplary flow cytometers that can be used in the methods provided herein, in addition to any conventional flow cytometer, can include flow cytometers that employ slow flow or signal integration times to allow sufficient time to register the
  • the signal integration time of the flow cytometer is between about 0.5 [ sec - 5000 [ sec; about 1 [ sec - 4000 [ sec; about 5 [ sec - 3000 [ sec; about 10 [ sec - 2000 [ sec; about 10 [ sec - 1000 [ sec; about 15 [ sec - 500 [ sec; about 15 [ sec - 100 [ sec; or about 20 [ sec - 50 ⁇
  • the flow cytometer can have a high laser power and high numerical aperture collection optics for improved sensitivity.
  • the flow cytometer can have a fluorescence resolution (R) of about 10 molecules FITC to about 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500 molecules of FITC, as measured in units of mean equivalent soluble fluorochromes (MESF).
  • R fluorescence resolution
  • the flow cytometer used in the methods provided herein can include instruments such as those described, for example, in Zhu et al. , ACS Nano 8: 10998-1 1006 (2014) and Zhang et al., Analytical Chemistry, 84:6421 -6428 (2012) .
  • Exemplary commercial flow cytometers for use in the methods provided herein include, for example, FACSCalibur (BD Biosciences) and CytoFlex (Beckman Coulter / Danaher Corporation) .
  • the sample flow rate setting of the flow cytometer can be the "Low” setting as designated in the instrument.
  • the sample flow rate setting can be "Medium” or "High,” as designated in the flow cytometer instrument.
  • An exemplary imaging flow cytometer for use in the methods provided herein is the I mageStream imaging flow cytometer from Amnis.
  • a sample containing particles that are membrane vesicles can be adjusted to a concentration suitable for optimal staining with the optically detectable label (surface area probe, such as di-8-ANEPPS, or molecular marker-specific probe, such as fluorescently labeled annexin V, cholera toxin B subunit or anti-CD61 ).
  • the optically detectable label surface area probe, such as di-8-ANEPPS, or molecular marker-specific probe, such as fluorescently labeled annexin V, cholera toxin B subunit or anti-CD61 .
  • the sample can be serially diluted whereby a final particle concentration of between about 1 -5 x 10 8 to about 1 -5 x 10 10 particles / ⁇ _ (an average of about 1 -5 x 10 9 particles / ⁇ _) is obtained.
  • the serial dilution of the sample can be from about 2-fold to about 100,000-fold or more, depending on the sample.
  • the dilution to reach a desired staining optimum is high, about 100-fold to about 10,000-fold, generally about 200-fold, due to the presence of interfering proteins in the plasma that bind to the label (e.g. , the fluorescent label di-8- ANEPPS).
  • the sample is cerebrospinal fluid (CSF), urine or saliva, the dilution factor is less, about 5-fold to about 100-fold, generally about 20-fold, due to lower amounts of interfering protein in these samples.
  • the samples are diluted in an isotonic buffer, such as PBS or Hanks Balanced Salt Solution (HBSS) and a small amount of surfactant (between about 0.005% to about 0.1 %, in some embodiments about 0.01 %) is added to facilitate incorporation of the surface area probe into the particle.
  • an isotonic buffer such as PBS or Hanks Balanced Salt Solution (HBSS)
  • HBSS Hanks Balanced Salt Solution
  • surfactant is a poloxamer and in some embodiments, the poloxamer is Pluronic- 127.
  • a surface area probe or volume probe is used to analyze the particles.
  • the surface area probe or volume probe is added at a concentration, generally between about 1 nM to 1 ⁇ , that achieves stoichiometric staining of the particle surface area or volume, respectively, and provides a measurable optical signal that is proportional to the surface area or volume, respectively.
  • concentration of added probe is about 500 nM.
  • a molecular marker-specific probe is used to analyze the particles.
  • the molecular marker-specific probe can be any ligand that can bind to or otherwise associate with a molecular marker on the particle and includes an optically detectable label.
  • the molecular marker-specific probe is added at a concentration, generally between about 1 pM to 100 ⁇ , to produce stoichiometric binding or association with the molecular markers, whereby the optical signal is proportional to the number of molecular markers per particle.
  • concentration of added probe is about 25 - 100 nM .
  • more than one probe selected from a surface area probe, a volume probe or a molecular marker-specific probe can be added to the particles for analysis.
  • Staining of the particles with the probes is performed for an amount of time and at a temperature suitable for effective labeling and can be at ambient temperature (20 - 25 °C) or any suitable temperature above the phase transition of the particle being detected.
  • the incubation time can be from about 30 seconds to about 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours or longer, depending on the particles and the optically detectable labels.
  • the labels are di-8-ANEPPS (surface area probe) and fluorescently labeled annexin V (molecular marker-specific probe)
  • the incubation is for 1 hour at ambient temperature.
  • the sample is diluted to reduce background signals from free label and adjust the concentration of label-associated/bound particles for optimal measurement and analysis of the signal from the bound/associated label.
  • a plasma sample stained with 500 nM di-8-ANEPPS and 50 nm fluorescently labeled annexin V is diluted 1000-fold, prior to detection of the labels.
  • the stained particles are illuminated at a wavelength suitable for detection of the surface area/volume probe and/or the molecular marker-specific probe that is bound to or otherwise associated with the particles.
  • a 160 mW, 488 nm laser is used to illuminate the particles.
  • Exemplary wavelengths can include any suitable wavelength for detecting an optically detectable label.
  • Exemplary wavelengths for fluorescent surface area probes or volume probes are, e.g.
  • 457 nm, 472 nm, 488 nm, 492 nm (di-8-ANEPPS) and for fluorescent molecular marker-specific probes are, e.g. , 365 nm, 375 nm, 405 nm, 457 nm, 472 nm, 488 nm, 492 nm, 514 nm, 532 nm, 561 nm, 633 nm, 635-642 nm, 660 nm, 785 nm, 800 nm, 1064 nm.
  • the signals from the particle-associated optically detectable labels can be detected using any detector suitable for effective detection of the signals.
  • Exemplary optical elements for selecting and dispersing light can include, but are not limited to, band pass filters, dichroic mirrors or optical gratings for filtering or dispersing light onto a detector.
  • Exemplary detectors can include, but are not limited to, a photomultiplier tube (PMT), an avalanche photodiode, an avalanche photodiode array, a silicon- PMT, a hybrid PMT, a photodiode array, a charged cathode device (CCD), an electron multiplied CCD, a CMOS sensor detector, or any suitable photodetector.
  • PMT photomultiplier tube
  • CCD charged cathode device
  • CCD electron multiplied CCD
  • CMOS sensor detector or any suitable photodetector.
  • a 600 nm long pass filter and PMT can be used to detect di-8-ANEPPS and a 525/30 band pass filter and PMT can be used to detect fluorescently labeled annexin V.
  • the signal integration times are adjusted according to the brightness (signal intensity from the particle-associated optically detectable label) of the particles being analyzed and the type of flow cytometer used to perform the analysis. For example, when the particles are small, e.g. , nanoparticles, fewer numbers of fluorescent labels becomes associated with the particles, thereby producing dimmer particles. To maximize detection of fluorescent intensity in the measurement volume, signal integration times are extended for smaller particles.
  • the signal integration time of the flow cytometer is between about 0.5 [ sec - 5000 [ sec; about 500 [ sec - 5000 [ sec; about 1 [ sec - 4000 [ sec; about 5 [ sec - 3000 [ sec; about 10 [ sec - 2000 [ sec; about 10 [ sec - 1000 [ sec; about 15 [ sec - 500 [ sec; about 15 [ sec - 100 [ sec; about 20 ⁇ sec - 50 ⁇ sec; or about 10, 15, 20, 25, 30, 35, 40, 45 or 50 ⁇
  • the signal integration time is about 20 [ sec.
  • optical standard particle is a particle that can be used as a calibration standard in the optical methods provided herein, for determining characteristics such as the size (e.g. , diameter) and/or surface area/volume and/or quantity of particles for which these characteristics are heretofore unknown (and one or more of these characteristics are known for the optical standard particle).
  • an optical standard particle of known size, or a preparation of optical standard particles of a known distribution of sizes can be used to calibrate optical intensity in terms of surface area.
  • the diameters of the optical standard particles can be determined using NTA, and the equivalent surface area distribution calculated from the diameters.
  • the optical standard particles can then be labeled with a surface area probe or volume probe and the signal intensities of the probe associated with the particles can be measured and plotted against the surface areas of the particles, thereby obtaining a correlation between optical intensity and surface area or volume, respectively, that can be used to determine the surface area or volume, based on measured optical intensity, of a particle of unknown size.
  • an optical standard particle having a known number of molecular marker molecules can be used, or a preparation of optical standard particles having a distribution of different known numbers of molecular marker molecules associated with the particles can be used.
  • the numbers of molecular marker molecules on the optical standard particles can be determined by labeling the particles with molecular marker-specific probe, then calibrating their intensity against an external standard.
  • the optical intensity is fluorescence intensity
  • the external standard can be the fluorophore FITC (fluorescein isothiocyanate) or PE (phycoerythrin) and the intensity is expressed as mean equivalent soluble fluorochromes (MESF).
  • An optical standard particle as used herein, also can be a lipid-containing particle including, but not limited to, a liposome, EV or other lipid-containing vesicle, containing a known amount of lipid. Such particles can be stained in one or more known amounts using one or more known amounts of probe, thereby obtaining one or more staining solutions containing optical standard particles associated with probe at one or more known probe to lipid ratios.
  • These staining solutions containing optical standard particles stained at known probe to lipid ratios can then be used, for example, to obtain a correlation between the values of the ratios and the detection of a spectral shift, as determined by a change in optical wavelength at which the maximum optical intensity of the stained optical standard particles is detected, or as determined by the ratio of optical intensities at two optical wavelengths.
  • a probe to lipid ratio at which a spectral shift is detected, or a change in ratio of optical intensities is determined, is identified as a probe to lipid ratio that approaches or is at saturation.
  • the probe to lipid ratio of the optical standard particle that is identified as a probe to lipid ratio that approaches or is at saturation can be used to determine the amount of probe to be added to the sample containing particles to be analyzed, whereby the resulting probe to lipid ratio of the particles to be analyzed approaches or is at saturation.
  • the correlation can be predetermined, or the sample containing particles to be analyzed can be spiked with one or more known amounts of the optical standard particle to determine the amount of probe that results in a probe to lipid ratio that approaches or is at saturation.
  • All lipids including: L-a-phosphatidylcholine (Egg, Chicken) [PC]; L-a- phosphatidylethanolamine (Egg, Chicken) [PE]; GM 1 ganglioside (Brain, Ovine- Ammonium Salt) [GM 1 ]; L-a-phosphatidylserine (Brain, Porcine) (sodium salt) [PS]; Sphingomyelin (Brain, Porcine); Cholesterol (Ovine, wool >98%) ; 1 ,2-dipalmitoyl-sn- glycero-3-phosphoethanolamine-N-(biotinyl) (sodium salt) [PE-b]; and 1 -palmitoyl-2- (dipyrrometheneboron difluoride)undecanoyl-sn-glycero-3-phosphocholine [Top Fluor PC] were purchased from Avanti Polar Lipids.
  • Di-8-ANEPPS (4-[2-[6-(Dioctylamino)- 2-naphthalenyl]ethenyl]- 1 -(3-sulfopropyl)-pyridinium) was from Biotium; DiOC6(3) iodide (3,3'-Dihexyloxacarbocyanine iodide) and PKH67 were from Sigma. Nile Red fluorescent beads (0.53 ⁇ and 0.1 1 ⁇ ) were from Spherotech.
  • Recombinant Annexin V and hamster anti-ratCD61 were labeled with Dylight488-succinimidyl ester (Pierce) and the F/P ratios determined following the manufacturer's instructions.
  • PPAK D-Phe-L-Pro-L-Arg-chloromethyl ketone
  • PBS Phosphate buffered saline
  • Mg ++ Mg ++ was from Corning. All other reagents were from Sigma- Aldrich. Glass tubes were from Fisher and microfuge tube were from Axygen. All buffers, diluents, and sheath fluid was filtered through a 0.1 urn pore diameter filter (Pall Corporation) before use.
  • Lipids were dissolved in chloroform at concentrations varying between 2.5- 100 mg/ml.
  • the composition (mole percent) of lipid vesicles was 47.9% PC, 16% PE, 15% sphingomyelin, 13% cholesterol, 6% PS, 1 % GM 1 , and 1 % PE-b.
  • the lipids, prepared at the described molar ratio at a final amount of 10 ⁇ total lipid, were added to a 12 x 75mm test tube.
  • the chloroform solvent was slowly evaporated in a fume hood under a light stream of N 2 gas, until a thin lipid film remained around the bottom of the tube.
  • the thin lipid film was then hydrated with 1 mL of 0.1 ⁇ filtered PBS (no Ca ++ , Mg ++ ) buffer and vortexed vigorously.
  • the resulting multi-lamellar vesicles (MLVs) were subjected to three freeze/thaw cycles alternating between an ethanol bath on dry ice and a 40°C warm water bath. Following the freeze/thaw cycles the solution was incubated for an additional 60 minutes, with vortexing every 15 minutes. MLVs were aliquoted into 100 ⁇ volumes and stored at -20°C.
  • MLVs were extruded through a LipsoFast extrusion device (Avestin) fitted with Nucleopore polycarbonate filter membrane with average pore diameters of 0.4 ⁇ , 0.2 ⁇ , 0.1 ⁇ , 0.08 ⁇ , and 0.05 ⁇ (GE Water & Process Technology) for a total of 21 passes (an odd number so as to minimize contamination with un-extruded particles in final syringe).
  • Liposomes were collected in 1 .5 mL microfuge tubes, aliquoted, and stored for up to several weeks at 4°C.
  • mice Female Sprague Dawley rats (1 1 weeks of age) were obtained from Charles River Laboratories (Wilmington, MA) and cared for in accordance with the Guide for the Care and Use of Laboratory Animals, 8th Edition (26). All animals were determined to be pathogen free by Charles River Laboratories assessment plus profile testing. Animals were individually housed at an AAALAC, Intl-accredited facility in non-sterile ventilated polycarbonate micro-isolator cages on corncob bedding. All research protocols were approved by Amgen Inc. (Seattle, WA) Institutional Animal Care and Use Committee. Animals had ad libitum access to pelleted feed (2020X Teklad; Harlan Laboratories Inc.
  • Liposomes were diluted in filtered DPBS and loaded into the chamber of a Nanosight LM-20 equipped with a 532 nm laser and a CCD (charged cathode device) camera.
  • the optimal camera level (setting: 15) and threshold (setting: 2) were established in preliminary experiments. Five movies of 60 seconds each were recorded and analyzed for each sample using the NanoSight software. Average histograms and mean diameters are reported.
  • Samples were analyzed on a FACSCalibur (BD Biosciences) equipped with stock lasers, filters, and detectors and using the "Low" sample flow rate setting. Samples also were analyzed using a flow cytometer of higher sensitivity constructed using components from commercial instruments.
  • the constructed flow cytometer contained an optical bench (flow cell, excitation laser beam shaping optics, forward angle obscuration bar, orthogonal collection optics, and optical relay fibers) from a FACSCanto flow cytometer (BD Biosciences, San Jose, CA) , a 488 nm laser (200 mW, Sapphire, Coherent), and a multi-PMT detector assembly from a Beckman Coulter Elite cell sorter.
  • Green (DyLight 488) and red (di-8-ANEPPS) fluorescence was collected through a 525/40 bandpass or a 600 LP, respectively.
  • Sheath and sample flow was provided by continuous flow pumps (Milligat, GlobalFIA, Fox Island, WA) at rates of 20 ⁇ / ⁇ and 0.02 ⁇ _ /sec respectively, which gave a transit time (pulse width) of ⁇ 20 [ sec through the probe volume (about 10x longer than a conventional flow cytometer).
  • PMTs and photodiodes digitized signal pulses from the analogue detectors (PMTs and photodiodes) and recorded pulse height and area from each channel, plus pulse width from the trigger channel.
  • PMTs and photodiodes analogue detectors
  • Some histogram data was analyzed using the Kolmogorov-Smirnov (KS) test (27).
  • Samples were serially diluted into 100 ⁇ _ of 0.1 ⁇ filtered HEPES buffered saline (HBS; 150 mM NaCI, 10 mM HEPES pH 7.4) containing 500 nM di-8-ANEPPS, 0.01 % Pluronic-127, 5 mM CaCI 2 and 20 ⁇ PPAK in a row of a 96 well plate, stained for at least 60 minutes, then diluted 1 :800 in PBS and analyzed by flow cytometry to determine the dilution of sample that gave optimal staining.
  • HBS HEPES buffered saline
  • EV preps were diluted in staining buffer as above, 50 nM of Dyl_ight488-labeled surface marker added, and the sample stained for at least 60 minutes (determined in preliminary experiments) at ambient temperature, followed by dilution and analysis by HSFC.
  • the di-8 intensity was plotted against surface area (calculated from NTA diameter estimates, assuming sphericity) at frequency intervals of 0.1 .
  • the slope and intercept of this line was used to convert channel number to surface area (nm 2 ), and then diameter was calculated with the assumption that the vesicles are spherical.
  • fluorophores practical use of MESF values. JOURNAL OF RESEARCH-NATIONAL INSTITUTE OF STANDARDS AND TECHNOLOGY 2002; 107:339-354).
  • FIG. 1 shows side scatter and fluorescence intensity histograms of the particle measurements triggered on fluorescence (A-C) vs. scatter (D-F).
  • the trigger threshold required to minimize the background event rate threshold (dashed line) and the system background signal levels obtained using a software trigger (grey filled histogram) also are shown.
  • the histograms of Figure 1 show that the 0.53 ⁇ bead is readily detected using a side scatter trigger or a fluorescence trigger.
  • Single beads are clearly identified on both side scatter and Nile Red fluorescence channels, as are coincident events, which are approximately twice as bright.
  • the light scatter from the 0.1 1 ⁇ bead is much lower and as the trigger threshold is lowered, triggered events from particles in the sheath and sample fluids begin to dominate the histogram, even before the optical background of the system as determined by a software trigger is reached.
  • setting the fluorescence trigger threshold just above the software trigger background allows the dim (0.1 1 ⁇ ) fluorescent beads to be detected, while their light scatter is in background range.
  • lipophilic fluorescent probes were evaluated for their ability to stain membrane vesicles as surface area probes.
  • Surface area probes were assessed for their ability to bind saturably and stoichiometrically with membrane vesicles such that probe-associated fluorescence is proportional to surface area and the fluorescence is enhancement when bound to membrane relative to when the probe is free in solution.
  • Triton X-100 which solubilized the vesicles and reduced particle counts to background levels.
  • the extruded liposomes were found to be stable, with stored liposomes (4 months, 4 °C) retaining the same fluorescence staining properties as freshly extruded liposomes.
  • the results demonstrate that liposomes are attractive as possible reference particles to aid in the standardization of EV measurements.
  • Example 3 The results from Example 3 above suggested that di-8-ANEPPS intercalates into membrane vesicles in a stoichiometric manner, and thus could be useful to estimate vesicle surface area and, therefore, vesicle diameter.
  • di- 8-ANEPPS at a P/L ratio of > 0.1 was used to measure liposomes that had been prepared by extrusion through nucleopore filters with different pore sizes.
  • Figures 2A-D depict fluorescence intensity histograms of di-8-ANEPPS stained vesicles prepared by extrusion through polycarbonate membrane filters with average pore sizes of 200 nm (A), 100 nm (B) , 80 nm (C) and 50 nm (D), as described in Example
  • Figures 2E-H depict nanoparticle diameter population histograms from NTA of the same vesicles, with the median diameter of each preparation indicated.
  • liposomes extruded through increasingly smaller pore sizes and stained with di-8-ANEPPS showed unimodal fluorescence intensity distributions with increasingly lower intensities.
  • these intensities ranged from ⁇ 25- 1000 mean equivalent PE-TexasRed molecules (MEPETR), determined using Spherotech Rainbow beads and the manufacturer's calibration values.
  • MEPETR PE-TexasRed molecules
  • NTA nanoparticle tracking analysis
  • Plasma from untreated rats was analyzed using the custom flow cytometer described in Example 1 .
  • Cell-free plasma was prepared from blood by low speed centrifugation (2x 2500xg, 10 minutes) to remove large cells and analyzed using NTA to determine total nanoparticle concentration and population size distribution.
  • Plasma from three different rats was measured by NTA ( Figures 4A-C) or by flow cytometry (vesicle flow cytometry or VFC; Figures 4D-F).
  • FIG. 4 shows NTA (4A-C) and flow cytometry (4D-F) population diameter distributions from the three representative rat plasma samples, estimated using liposomes as a reference particle as described above. Both NTA and flow cytometry reported unimodal particle populations, with a peak (mode) diameter of -120 nm. The correlation between NTA nanoparticle concentration estimates and flow cytometry vesicle concentration estimates for eight different animal samples are presented in Figure 4G.
  • NTA which measures particles with detectable light scatter
  • flow cytometry which measures particles with detectable fluorescence
  • platelet rich plasma (PRP) from rats were prepared and stimulated with the ionophore A23187, which is known to induce platelet EV release. After treatment, samples were centrifuged as above to remove residual cells and platelets and stained with di-8-ANEPPS as a surface area probe, followed by staining with either Dylight488-Annexin V or -CD61 as a membrane molecular marker-specific probe.
  • CD61 is a component of the ⁇ 2 ⁇ 3 integrin complex found in high abundance on platelets, while annexin V binds to exposed phosphatidylserine (PS) on the outer leaflet of the vesicle membrane.
  • Figure 5 depicts the fluorescence measurement of EV surface markers from control plasma (5A, 5B) or ionophore-treated platelet-rich plasma (5C, 5D).
  • EVs stained with both di-8-ANEPPS and Dylight 488-Annexin V show a clearly resolved population of EVs, with annexin V binding corresponding to ⁇ 28% of total EVs with a median brightness equivalent to ⁇ 6400 MESF of fluorescein, as well as a population that was annexin V negative (Figure 5A).
  • ionophore >60% of the EVs were annexin V positive (Figure 5C).
  • CSF samples were stained using di-8-ANEPPS as a fluorescent surface area probe for the membranes of EVs in CSF, and Dyl_ight488- labeled surface markers (annexin V or anti-CD41) were used as fluorescent molecular marker-specific probes of the EVs.
  • the samples prepared for flow cytometry were analyzed using the custom constructed instrument described in Example 1 .
  • haemorrhage showed 2- to 7-fold increases in the concentration of EVs, compared to samples from normal subjects, with no notable differences in size, indicating that the EV concentrations could have diagnostic value.
  • the particle concentrations obtained by flow cytometry were lower than NTA (2.16 x 10 6 particles / ⁇ with flow cytometry, relative to 4.32 x 10 6 particles / ⁇ with NTA) and the mean size was larger (192 nm with flow cytometry, relative to 1 18 nm with NTA), indicating that NTA likely measures all scattering particles in the CSF while flow cytometry measures membranous nanoparticles.
  • the measured intensities of the fluorescent molecular marker-specific probes associated with the EVs revealed variable annexin V - associated EV fractions, with some showing a significant fraction of CD41 - associated EVs.
  • fluorescence measurement based flow cytometry could characterize the EVs in CSF with high sensitivity compared to NTA, with flow cytometry providing fluorescent antibody based speciation of EVs.
  • a set of 0.45 urn antibody capture beads that each capture about 5000 antibody molecules were developed as positive control nanobeads (optical standard particles) and BSA coated beads were developed as negative control nanobeads (optical standard particles).
  • 450 nm polystyrene beads were coated with either anti-lambda IgG (positive control) or BSA (negative control).
  • Such beads can serve as a spectral reference for use in compensation or spectral unmixing, to obtain a more accurate detection and/or quantitation of the fluorescence intensity of nanoparticles (e.g. , EVs) of interest.
  • Figure 6 depicts histograms from a sample containing both the positive and negative control beads stained with a Dyl_ight488-conjugated antibody.
  • the antibody capture beads can be used as compensation standards for flow cytometry measurements of particle, including nanoparticle, surface markers or in multispectral measurements of particle, including nanoparticle (e.g. , EV) surface molecular markers by capturing antibodies labeled with multiple different
  • Such a panel of antibody capture beads can provide efficient analyses of multiple nanoparticle-associated molecular markers, while serving as spectral reference particles to correct for spectral mixing between the different.
  • the spectral data stream can have the background Rayleigh and Raman scatter spectra subtracted in real time and data can be analyzed in the conventional mode, where signals from various spectral bands are plotted as intensity histograms, or as a hyperspectral data set that allows spectral approaches that can produce higher resolution measurements.
  • Example 8 Characterized Liposomes for Detection and/or Sizing of Vesicle- Associated Molecular Markers.
  • Liposomes i.e. , synthetic lipid vesicles, were prepared with defined compositions and nanoparticle size distributions that can be measured by independent methods (NTA, RPS, TEM), to serve as reference particles for the analysis of EVs.
  • the liposomes were found to be stable for months at 4 °C.
  • Figure 7 depicts flow cytometry bivariate histograms showing di-8-ANEPPS vs. Brilliant Violet-conjugated Annexin V fluorescence of liposomes with or without phosphatidylserine (PS) , which binds to annexin V.
  • PS phosphatidylserine
  • Sub-micron sized silica lipospheres bearing a supported lipid bilayer were developed for detection by light scatter or for staining by surface area probe membrane dyes such as Di-8-ANEPPS and annexin V.
  • 500 nm silica nanospheres were coated with a lipid bilayer, generating silica lipospheres.
  • the 500 nm silica beads (nanospheres) were obtained from Duke Scientific (#8050) and sonicated to disperse the beads. 25 ⁇ _ of a stock solution of the beads (3 x 10 11 bead particles/ml) was pipetted into 1 ml_ of 10 mM HEPES buffer pH 7.3, 150 mM NaCI.
  • silica liposphere preparation was stored at 4 °C, at a concentration of 1 x 10 9 bead particles/ml.
  • the silica lipospheres can serve as both positive staining controls and spectral compensation reference particles.
  • Presented in Figure 8 are data from lipospheres stained with either Di-8-ANEPPS or Dyl_ight488-annexin V and analyzed on the custom flow cytometer constructed as described in Example 1 , using side scatter as a detection trigger.
  • Figure 8 shows two distinct liposphere populations, the population stained with di-8-ANEPPS showing enhanced di-8-ANEPPS fluorescence and the population stained with Dyl_ight488-Annexin V showing enhanced annexin V fluorescence. The results demonstrate that both dyes are capable of binding to the lipospheres, thereby validating their use as reference particles.
  • Example 10 Determining Membrane Dye Saturation using Fluorescence Spectral Shift
  • Figures 9A and 9B are fluorescence spectra of bulk suspensions of di-8-ANEPPS (500 nM) in buffer alone (HBS; 150 mM NaCI, 10 mM HEPES pH 7.4) or buffer plus two concentrations of synthetic lipid vesicles (50 uM and 3 uM , prepared as in Example 1 above) to produce two different probe to lipid ratios (0.01 and 0.16, respectively) .
  • HBS buffer alone
  • buffer plus two concentrations of synthetic lipid vesicles 50 uM and 3 uM , prepared as in Example 1 above
  • the probe to lipid ratio is higher and the fluorescence emission maximum is shifted shifts towards the red.
  • This spectral shift can be expressed as the ratio of fluorescence intensity at 690 nm to the fluorescence intensity at 610 nm, and can be used to monitor the probe as it approaches saturation in the membrane.
  • Figure 9B is a normalized representation of the measurements depicted in Figure 9A.
  • Figure 9C is the ratio of intensities at 690 to 610 nm measured at several different probe to lipid ratios. The measurements depicted in Figures 9A-C were performed in a fluorimeter on bulk solutions in containers, such as cuvettes.
  • the spectral shift also can be monitored by flow cytometry, using the ratio of fluorescence intensity measured through a 690/50 nm band pass filter to the intensity measured through a 610/20 nm bandpass filter.
  • Flow cytometry permits the analysis of individual particles / vesicles.
  • Figure 9D are histograms of the population distributions of the ratio of intensities of the synthetic vesicles measured through the 690/50 nm and 610/20 nm filters (690/610 ratio) , for high (0.16) and low (0.01 ) probe to lipid ratios.
  • the vesicles stained with the higher probe to lipid ratio have a higher 690/610 ratio, which indicates a higher degree of saturation.
  • Figure 9E presents the ratio of intensities at 690 to 610 nm measured by flow cytometry of synthetic vesicle preparations having several different probe to lipid ratios.
  • monitoring the 690/610 ratio provides a means to measure when probe saturation is approached in synthetic vesicle staining as well staining of biological vesicular preparations.
  • Example 11 Detection of Vesicles and Measurement of their Light Scatter
  • Flow cytometry of cells generally employs light scatter to trigger detection, and most examples of vesicle measurements by flow cytometry also use this approach, which presents a number of difficulties including, but not limited to: 1 ) the very dim light scatter signals produced by vesicle owing to their small size and low refractive index; 2) background light scatter from particles in sample, reagents, buffers, and sheath fluids as well as scatter from the flow cell and other optical components; 3) differentiation between the dim scatter from vesicles as discussed in 1 ) and the various sources of background as discussed in 2) ; and 4) interpreting light scatter intensity given its complex dependence on illumination wavelength, particle size
  • the fluorescence-based detection approach which uses a fluorogenic membrane probe, obviates many of these issues by selectively detecting membrane vesicles with fluorescence intensity that is proportional to vesicle size (surface area) and rendering light scatter a more useful measurement parameter.
  • Example 12 Multimar er analysis of EVs in Human Plasma
  • Vesicles in complex biological fluids such as plasma are heterogeneous, originating from different cell types and from different compartments within cells.
  • PECy7-annexin V (a marker of phosphatidyl serine), PE-anti-CD41 (a marker of CD41 on platelets), and APC-anti-CD235 (a marker of CD235 on erythrocytes).
  • Annexin V has a specific binding affinity for phosphatidyl serine (PS), which is a surface molecular marker of many cell-derived EVs, membrane vesicles and liposomes.
  • PS phosphatidyl serine
  • Synthetic liposomes (prepared as in Example 1 above) bearing phosphatidylserine were also stained using PECy7-annexin V as a positive control and PE-anti-CD41 and APC-anti-CD235 as negative controls, since they do not contain the CD41 and CD235 antigens.
  • Intensity calibration beads for PE and APC were used to calibrate these signals in units of mean equivalent soluble
  • Figure 1 1A is the population size distribution of the synthetic liposomes as estimated from di-8-ANEPPS staining intensity and bivariate histograms of diameter, versus fluorescence intensity of the three different fluorescence-labeled reagents.
  • the liposomes showed strong staining for annexin V (PECy7 Intensity) and low levels of background from the PE-anti-CD41 (PE Intensity) and APC-anti- CD235 (APC Intensity), likely resulting from antibody aggregates.
  • Treatment of the sample with the detergent Triton X- 100 (0.05%) eliminates the liposomes, while the antibody aggregates and other non-membrane background events remain (Figure 1 1 B).
  • Figure 1 1 C is the population size distribution of EVs in plasma as estimated from di-8-ANEPPS staining intensity and bivariate histograms of diameter, versus fluorescence intensity of the three different fluorescence-labeled reagents. Subsets of EVs showed staining for CD41 (PE Intensity) and annexin V (PE-Cy7 Intensity) . When this sample was treated with 0.05% Triton X- 100, the EVs were eliminated leaving behind the detergent-insoluble background particles Figure 1 1 D) .
  • this method allows the selective detection of membrane vesicles, estimation of their size, and measurement of surface molecule markers directly in plasma.
  • Example 13 Examples of certain non-limiting embodiments
  • a method of analyzing particles in a sample comprising:
  • the one or more optically detectable labels comprise a surface area probe or volume probe, wherein the surface area probe or volume probe interacts with the particles stoichiometrically with respect to particle surface area or volume, respectively, thereby forming particles comprising particle- associated surface area probe or volume probe, wherein the optical signal from the particle-associated surface area probe or volume probe is proportional to the surface area or volume, respectively, of the particle, and/or
  • the one or more optically detectable labels comprise a molecular marker-specific probe, wherein the molecular marker-specific probe interacts with a molecular marker of the particle stoichiometrically with respect to the number of molecules of the molecular marker that are associated with the particle, thereby forming particles comprising particle-associated molecular marker-specific probe, wherein the optical signal from the particle-associated molecular marker-specific probe is proportional to the number of molecules of molecular marker associated with the particle; and
  • A5 The method of any one of embodiments A1 to A4, wherein at least one particle in the sample comprises a size of between about 50 nm to about 150 nm in diameter.
  • A6 The method of any one of embodiments A1 to A5, wherein the particles in the sample comprise a size range of between about 10 nm to about 500 nm in diameter.
  • A7 The method of any one of embodiments A1 to A6, wherein the particles in the sample comprise a size range of between about 50 nm to about 200 nm in diameter.
  • A8 The method of any one of embodiments A1 to A7, wherein the particles in the sample comprise a size range of between about 50 nm to about 150 nm in diameter.
  • A9 The method of any of embodiments A1 to A8, wherein prior to (a), the concentration of the particles in the sample is, or is adjusted to, between about 1 x 10 6 particles/ ⁇ . to about 1 x 10 12 particles ⁇ L.
  • A13 The method of embodiment A1 1 , wherein the isotonic buffer is phosphate buffered saline (PBS), Hanks balanced salt solution (HBSS) or HEPES buffered saline.
  • PBS phosphate buffered saline
  • HBSS Hanks balanced salt solution
  • HEPES HEPES buffered saline
  • A15 The method of any of embodiments A1 to A14, wherein the concentration of the surfactant in the staining solution is between about 0.005% to about 0.1 %.
  • A16 The method of any of embodiments A1 to A15, wherein analyzing the particles in the sample comprises detecting the particles in the sample.
  • A16.1 The method of any of embodiments A1 to A16, wherein analyzing the particles in the sample comprises determining the surface area or volume of the particle based on the detected optical signal of the surface area probe or volume probe, respectively.
  • A16.2 The method of embodiment A16.1 , further comprising determining the size of the particle based on the surface area or volume.
  • analyzing the particles in the sample comprises determining the type and/or number of molecular markers associated with the particle based on the detected optical signal of the molecular marker-associated probe.
  • A17.1 The method of embodiment A17, further comprising identifying and/or quantifying the particle based on the type and/or number of molecular markers associated with the particle.
  • A20 The method of embodiment A18 or A19, wherein the fluorescent label is a fluorophore, a tandem conjugate between more than one fluorophore, a fluorescent polymer, a fluorescent protein, or a fluorophore conjugated to a molecule that interacts with the particle.
  • the fluorescent label is a fluorophore, a tandem conjugate between more than one fluorophore, a fluorescent polymer, a fluorescent protein, or a fluorophore conjugated to a molecule that interacts with the particle.
  • A22 The method of embodiment A21 , wherein the molecule that interacts with the particle is a protein, an antibody, a lectin, a peptide, a nucleic acid, a carbohydrate or a glycan.
  • A23 The method of any of embodiments A1 to A22, wherein at least one particle comprises a lipid bilayer.
  • A24. The method of embodiment A23, wherein the particle comprising a lipid bilayer is a liposome or an extracellular vesicle.
  • A25 The method of any of embodiments A18 to A24, wherein detection of the optically detectable label is by fluorescence spectroscopy, fluorescence imaging, or flow cytometry.
  • A27 The method of embodiment A26, wherein the particle is a liposome or an extracellular vesicle.
  • A30 The method of any of embodiments A27 to A29, wherein the ratio of the amount surface area probe (P) relative to the amount of lipid (L) in the particle, P/L, is adjusted whereby the surface area probe interacts with the particles
  • A32.1 The method of embodiment A32, wherein the protein is selected from among annexin V, cholera toxin B-subunit,anti-CD61 , anti-CD171 , anti-CD325, anti-CD130, anti-GLAST, anti-EGFRvlll , anti-EGFR, anti-CD133, anti-CD15, anti-CD63, anti-CD9, anti-CD41 , anti-CD235, anti-CD54 and anti-CD45.
  • fluorophore conjugated to the protein conjugates is selected from among Dylight488, a Brilliant Violet dye, Pacific Blue, Chrome Orange, Brilliant Blue 515, PE, rhodamine, FITC, PE-Cy5.5, PE-Cy7, APC, Alexa647, APC-Alexa700 and APC-Alexa750.
  • A34 The method of any of embodiments A1 to A33, wherein physical separation or isolation of the particles comprises washing of the particles.
  • A35 The method of any of embodiments A1 to A34, wherein physical separation or isolation of the particles comprises centrifugation or ultracentrifugation of the particles.
  • A36 The method of any of embodiments A26 to A35, wherein the flow cytometer has a configuration whereby light is collected from both sides of the flow cell.
  • A37 The method of any of embodiments A26 to A36, wherein the detection range of the flow cytometer is between about 500 fluorescent molecules per particle to about 5000 fluorescent molecules per particle.
  • the particle is an extracellular vesicle
  • the type of molecular marker associated with the extracellular vesicle is determined.
  • A40.1 The method of embodiment A40, further comprising identifying the cell and/or tissue of origin of the extracellular vesicle based on the type of molecular marker associated with the extracellular vesicle.
  • A41 The method of any of embodiments A1 to A40, wherein the sample comprises a plurality of particles and the method is for determining the size distribution of the plurality of particles.
  • A43 The method of any of embodiments A1 to A42, wherein the interaction of the surface area or volume probe and/or the molecular marker-specific probe with the particle is saturable, whereby the optical signal from the surface area probe or volume probe is proportional to the surface area or volume, respectively, of the particle and/or the optical signal from the molecular marker-specific probe is proportional to the number of molecules of molecular marker associated with the particle.
  • B1. A method of detecting, identifying, quantifying and/or determining the size of at least a first nanoparticle species in a sample comprising at two distinct nanoparticle species, the method comprising:
  • the molecular marker-specific probe interacts with a molecular marker of at least the first nanoparticle species stoichiometrically with respect to the number of molecules of the molecular marker that are associated with the nanoparticle, thereby forming particles comprising particle-associated molecular marker-specific probe, wherein the optical signal from the particle-associated molecular marker-specific probe is proportional to the number of molecules of the molecular marker that are associated with the first nanoparticle species;
  • probe or volume probe obtained in (c), determining the surface area or volume, respectively, of at least the first nanoparticle species, thereby detecting and/or determining the size of at least the first nanoparticle species in the sample; and/or (e) based on the optical intensity of the particle-associated molecular marker- specific probe obtained in (c) , determining the type and/or number of molecular markers associated with at least the first nanoparticle species, thereby detecting, identifying and/or quantifying at least the first nanoparticle species in the sample.
  • B12.1 The method of embodiment B12, wherein the protein is selected from among annexin V, cholera toxin B-subunit,anti-CD61 , anti-CD171 , anti-CD325, anti-CD130, anti-GLAST, anti-EGFRvlll , anti-EGFR, anti-CD133, anti-CD15, anti-CD63, anti-CD9, anti-CD41 , anti-CD235, anti-CD54 and anti-CD45.
  • the protein is selected from among annexin V, cholera toxin B-subunit,anti-CD61 , anti-CD171 , anti-CD325, anti-CD130, anti-GLAST, anti-EGFRvlll , anti-EGFR, anti-CD133, anti-CD15, anti-CD63, anti-CD9, anti-CD41 , anti-CD235, anti-CD54 and anti-CD45.
  • fluorophore conjugated to the protein conjugates is selected from among Dylight488, a Brilliant Violet dye, Pacific Blue, Chrome Orange, Brilliant Blue 515, PE, rhodamine, FITC, PE-Cy5.5, PE-Cy7, APC, Alexa647, APC-Alexa700 and APC-Alexa750.
  • optical standard particle corresponding to at least the first nanoparticle species
  • the optical standard particle comprises an optically detectable label that is the surface area or volume probe that interacts with the first nanoparticle species in (a) or the optical standard particle comprises an optically detectable label that is the molecular marker-specific probe that interacts with the first nanoparticle species in (b);
  • optical standard particle comprising the optically detectable label further comprises an antibody, a liposome or a silica particle.
  • identifying the type or identity of the first nanoparticle species comprises determining the cellular origin of the extracellular vesicle.
  • extracellular vesicle is a cancer cell.
  • optical standard particle comprising the optically detectable label comprises a silica particle in association with a lipid bilayer.
  • optical standard particle comprises an optically detectable label that is a surface area probe.
  • B34 The method of embodiment B33, wherein the surface area probe is a fluorophore selected from among di-8-ANEPPS, di-4-ANEPPS, a carbocyanine dye and a PKH dye.
  • B35 The method of embodiment B34, wherein the surface area probe is di-8- ANEPPS.
  • a method of determining the size of a nanoparticle of interest in a sample using an optically detectable label comprising:
  • the preparation of nanoparticles is contacted with the optically detectable label used in (a) ;
  • the optically detectable label interacts stoichiometrically with each of the nanoparticles of the preparation, whereby nanoparticles comprising nanoparticle- associated optically detectable label are obtained, wherein the optical signal from each nanoparticle-associated optically detectable label is proportional to the surface area or volume of its corresponding associated nanoparticle;
  • optically detectable label comprises the surface area probe or volume probe in (a) , wherein the surface area probe or volume probe interacts with the nanoparticles stoichiometrically with respect to nanoparticle surface area or volume, respectively, whereby the optical signal from the optically detectable label is proportional to the surface area or volume, respectively, of each nanoparticle in the preparation;
  • the optically detectable label interacts with the nanoparticle of interest and with the preparation of nanoparticles, whereby a nanoparticle of interest comprising nanoparticle of interest-associated optically detectable label and nanoparticles comprising nanoparticle-associated optically detectable label are obtained;
  • optical signal intensities are obtained from the nanoparticle of interest- associated optically detectable label and the nanoparticle-associated optically detectable label in (b);
  • the size of the nanoparticle of interest is determined in (d) based on the predetermined correlation obtained according to (c) and based on the optical signal intensities obtained from the nanoparticle of interest-associated optically detectable label and the nanoparticle-associated optically detectable label in (b).
  • a method of identifying and/or quantifying a nanoparticle of interest in a sample using an optically detectable label comprising:
  • the optically detectable label interacts stoichiometrically with each of the nanoparticles of the preparation, whereby nanoparticles comprising nanoparticle- associated optically detectable label are obtained, wherein the optical signal from each nanoparticle-associated optically detectable label is proportional to number of molecules of the molecular marker on the corresponding associated nanoparticle;
  • the optical signal intensity of each nanoparticle-associated optically detectable label obtained in (iii) is correlated with the identity and/or quantity of its corresponding associated nanoparticle; and (d) based on the predetermined correlation obtained in (c) , and based on the optical signal intensity obtained in (b), identifying and/or quantifying the nanoparticle of interest.
  • optically detectable label comprises the molecular marker-specific probe in (a), wherein the molecular marker-specific probe interacts with the nanoparticles stoichiometrically with respect to the number of molecules of molecular marker associated with each nanoparticle of them preparation, whereby the optical signal from the optically detectable label is proportional to the number of molecules of molecular marker associated with each nanoparticle of the preparation;
  • optical signal intensities are obtained from the nanoparticle of interest- associated optically detectable label and the nanoparticle-associated optically detectable label in (b);
  • the nanoparticle of interest is identified and/or quantified in (d) based on the predetermined correlation obtained according to (c) and based on the optical signal intensities obtained from the nanoparticle of interest-associated optically detectable label and the nanoparticle-associated optically detectable label in (b) .
  • a preparation of optical standard particles comprising a silica particle and a lipid bilayer in association with the silica particle, wherein the preparation has a distribution of optical standard particle sizes between about 10 nm to about 900 nm.
  • a optical standard particle comprising a silica particle and a lipid bilayer in association with the silica particle.
  • optical standard particle of embodiment E5 or E6, wherein the size of the optical standard particle is about 50 nm to about 200 nm.
  • optical standard particle of embodiment E7, wherein the size of the optical standard particle is about 100 nm to about 150 nm.
  • the biological fluid comprises blood, plasma, serum, urine, saliva, seminal fluid, lavages, cervical fluid, cervicovaginal fluid, cerebrospinal fluid, vaginal fluid , breast fluid , breast milk, synovial fluid, semen, seminal fluid , sputum, cerebral spinal fluid, tears, mucus, interstitial fluid, follicular fluid, amniotic fluid, aqueous humor, vitreous humor, peritoneal fluid, ascites, sweat, lymphatic fluid, lung sputum or combinations, fractions or components thereof.
  • the biological fluid comprises cerebrospinal fluid.
  • F8 The method of embodiment F6 or F7, wherein the cell or tissue is selected from among liver, lung, spleen, pancreas, colon, skin, bladder, eye, brain, esophagus, head, neck, ovary, testes, prostate, placenta, epithelium, endothelium, adipocyte, kidney, heart, muscle, blood and combinations thereof.
  • F1 1 The method of embodiment F 10, wherein the biological fluid is saliva, urine or serum.
  • F15 The method of embodiment F14, wherein the brain cancer is glioblastoma.
  • the first staining solution comprises one or more lipid-containing particles comprising a ratio of probe to lipid that is approaching saturation or has saturated. H1 .
  • the method of embodiment HO further comprising:
  • the maximum optical signal wavelength of the staining solution is about the same as the maximum optical signal wavelength of a third staining solution comprising a higher ratio of probe concentration to sample amount and/or a higher ratio of probe to lipid.
  • a method of determining whether staining of a lipid-containing particle with an optically detectable label selected from among a surface area probe, a volume probe or a molecular marker-specific probe of a particle is approaching saturation or is saturated comprising:
  • H6 The method of any of embodiments HO to H4, wherein the difference between the first maximum optical wavelength and the second maximum optical wavelength is about 0.5 nm, 1 nm, 1 .5 nm, 2 nm, 2.5 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 12 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 21 nm, 22 nm, 23 nm, 24 nm, 25 nm, 26 nm, 27 nm, 28 nm, 29 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 n
  • the first staining solution comprises one or more lipid-containing particles comprising a ratio of probe to lipid that is approaching saturation or has saturated.
  • the staining solution comprises one or more lipid-containing particles comprising a ratio of probe to lipid that is approaching saturation or has saturated.
  • the ratio of optical signal intensity at the first optical wavelength relative to the optical signal intensity at the second optical wavelength of the staining solution is different from the ratio of optical signal intensity at the first optical wavelength relative to the optical signal intensity at the second optical wavelength of a second staining solution comprising a lower ratio of probe concentration to sample amount and/or a lower ratio of probe to lipid;
  • the ratio of optical signal intensity at the first optical wavelength relative to the optical signal intensity at the second optical wavelength of the staining solution is about the same as or greater than the ratio of optical signal intensity at the first optical wavelength relative to the optical signal intensity at the second optical wavelength of a second staining solution comprising a higher ratio of probe concentration to sample amount and/or a higher ratio of probe to lipid.
  • the probe to lipid ratio corresponding to the maximum ratio of optical signal intensity at the first optical wavelength relative to the optical signal intensity at the second optical wavelength is the probe to lipid ratio at which staining of the particle is approaching saturation or has saturated.
  • H15 The method of any of embodiments HO to H6, wherein the first maximum optical wavelength and/or the second maximum optical wavelength are in the range of between 350 to 950 nm, between 400-900 nm, or between 600 to 800 nm.
  • H16 The method of any of embodiments H7 to H14, wherein the first optical wavelength and/or the second optical wavelength are in the range of between 350 to 950 nm, between 400 to 900 nm, or between 600 to 800 nm.
  • At least one lipid- containing particle in the sample comprises a size of about 1000 nm, 950 nm, 900 nm, 850 nm, 800 nm, 750 nm, 700 nm, 650 nm, 600 nm, 550 nm or 500 nm or less in diameter.
  • H27 The method of any of embodiments H1 , H2, H4-H6, H8-H1 1 or H14-H26, wherein at least one optical standard particle in the sample comprises a size of about 1000 nm, 950 nm, 900 nm, 850 nm, 800 nm, 750 nm, 700 nm, 650 nm, 600 nm, 550 nm or 500 nm or less in diameter.
  • H28 The method of embodiment H27, wherein at least one particle in the sample comprises a size of between about 10 nm to about 200 nm in diameter.
  • H31 The method of any one of embodiments H1 , H2, H4-H6, H8-H1 1 or H14-H26, wherein the lipid-containing particles in the sample comprise a size range of between about 10 nm to about 500 nm in diameter.
  • H40 The method of any of embodiments H37 to H39, wherein the fluorescent label is conjugated to a molecule that interacts with the particle.
  • H41 The method of embodiment H40, wherein the molecule that interacts with the particle is a protein, an antibody, a lectin, a peptide, a nucleic acid, a carbohydrate or a glycan.
  • H44.1 The method of any of embodiments HO to H44, wherein the optical wavelength and/or intensity is obtained by analyzing the sample in bulk.
  • H50 The method of embodiment H49, wherein the surface area probe is di-8- ANEPPS.
  • H51 The method of any of embodiments H38 to H48, wherein the probe is a molecular marker-specific probe comprising a fluorophore conjugated to a protein
  • H52 The method of embodiment H51 , wherein the protein is selected from among annexin V, cholera toxin B-subunit, anti-CD61 , anti-CD171 , anti-CD325, anti-CD130, anti-GLAST, anti-EGFRvlll , anti-EGFR, anti-CD133, anti-CD15, anti-CD63, anti-CD9, anti-CD41 , anti-CD235, anti-CD54 and anti-CD45.
  • the protein is selected from among annexin V, cholera toxin B-subunit, anti-CD61 , anti-CD171 , anti-CD325, anti-CD130, anti-GLAST, anti-EGFRvlll , anti-EGFR, anti-CD133, anti-CD15, anti-CD63, anti-CD9, anti-CD41 , anti-CD235, anti-CD54 and anti-CD45.
  • optical wavelength and/or intensity is from fluorescence emission, fluorescence excitation, fluorescence absorbance, fluorescence anisotropy, fluorescence polarization, fluorescence lifetime, or a combination thereof.
  • a reagent can mean one or more reagents) unless it is contextually clear either one of the elements or more than one of the elements is described.
  • the term "about” as used herein refers to a value within 10% of the underlying parameter (i.e. , plus or minus 10%), and use of the term “about” at the beginning of a string of values modifies each of the values (i.e. , "about 1 , 2 and 3" refers to about 1 , about 2 and about 3).
  • a weight of "about 100 grams” can include weights between 90 grams and 1 10 grams.

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Abstract

Cette technologie concerne en partie des méthodes optiques pour analyser des particules, y compris des nanoparticules, ce qui permet de déterminer leur présence, leur identité, leur origine, leur taille et/ou leur nombre dans un échantillon d'intérêt.
PCT/US2016/046401 2015-08-11 2016-08-10 Analyse optique de particules et de vésicules WO2017027622A1 (fr)

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CN110780075A (zh) * 2018-07-27 2020-02-11 希森美康株式会社 生物体粒子测定用方法、装置及试剂盒、及非特异信号的检测方法
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KR20230021732A (ko) * 2020-06-09 2023-02-14 파티클 머슈어링 시스템즈, 인크. 입사 광과 조합된 산란 광을 통한 입자 검출
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US10429302B2 (en) 2019-10-01
US20200173923A1 (en) 2020-06-04

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